Treatment of cancer using anti CD19 chimeric antigen receptor

Abstract

To provide a composition for treating a disease associated with expression of surface antigen classification 19 protein (CD19), which is manipulated so as to express a chimeric antigen receptor (CAR) T-cell.SOLUTION: A composition contains a cell expressing a CAR molecule binding to CD19 to be used in combination with one or more kinase inhibitors in treatment of a mammal having a disease associated with expression of CD19, where the kinase inhibitor is selected from Bruton's tyrosine kinase (BTK) inhibitor, cyclin-dependent kinase 4 (CDK4) inhibitor, mTOR inhibitor or mitogen-activated protein kinase interaction kinase (MNK) inhibitor.SELECTED DRAWING: Figure 60

Description

[Technical Field]

[0001] This application claims priority to U.S. Application No. 61 / 976,396 (filed April 7, 2014), U.S. Application No. 62 / 007,309 (filed June 3, 2014), U.S. Application No. 62 / 036,493 (filed August 12, 2014), U.S. Application No. 62 / 076,238 (filed November 6, 2014), U.S. Application No. 62 / 087,888 (filed December 5, 2014), and U.S. Application No. 62 / 097,278 (filed December 29, 2014), the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing, which has been provided electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on April 6, 2015, is named N2067-7051WO_SL.txt and is 252,236 bytes in size.

[0003] FIELD OF THE INVENTION The present invention generally relates to the use of T cells engineered to express a chimeric antigen receptor (CAR), e.g., in combination with other agents such as, e.g., kinase inhibitors and / or cytokines, to treat diseases associated with expression of the Cluster of Differentiation 19 protein (CD19). [Background technology]

[0004] Background of the Invention Many patients with B-cell malignancies are incurable with standard therapies. Furthermore, traditional treatment options often have severe side effects. Although cancer immunotherapy has been attempted, several obstacles make achieving clinical efficacy an extremely difficult goal. Although hundreds of so-called tumor antigens have been identified, these are generally self-derived and therefore poorly immunogenic. Furthermore, tumors use several mechanisms to render themselves unsuitable for initiating and propagating immune attack.

[0005] Recent developments using chimeric antigen receptor (CAR)-modified autologous T cell (CART) therapy, which relies on redirecting T cells to appropriate cell surface molecules on cancer cells, such as B cell malignancies, have shown promising results in harnessing the power of the immune system to treat B cell malignancies and other cancers (see, e.g., Sadelain et al., Cancer Discovery 3:388-398 (2013)). Clinical results with murine-derived CART19 (i.e., "CTL019") have shown promise in establishing complete remissions in patients with CLL and pediatric ALL (e.g., Kalos et al., Sci Transl Med 3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368:1509-1518 (2013)). In addition to the ability of the chimeric antigen receptor on genetically modified T cells to recognize and destroy target cells, successful therapeutic T cell therapy requires the ability to proliferate and persist over time and further monitoring for escaped leukemia cells. The variable quality of T cells, whether the result of anergy, suppression, or exhaustion, affects the performance of CAR-transformed T cells, but those skilled in the art currently have limited control over this. To be effective, patient T cells transformed with CARs must persist and maintain the ability to proliferate in response to the CAR antigen. Using CART19 containing a mouse scFv, ALL patient T cells have been shown to be able to do this (see, e.g., Grupp et al., NEJM 368:1509-1518 (2013)). Summary of the Invention

[0006] Summary of the Invention The present invention relates, at least in part, to compositions and methods of treating disorders such as cancer (e.g., hematological cancer or other B-cell malignancies) using immune effector cells (e.g., T cells or NK cells) expressing a chimeric antigen receptor (CAR) molecule (e.g., a CAR that binds to a B-cell antigen, e.g., Cluster of Differentiation 19 protein (CD19) (e.g., OMIM Acc. No. 107265, Swiss Prot. Acc No. P15391). The compositions comprise, and the methods comprise, administration of immune effector cells (e.g., T cells or NK cells) expressing a B-cell targeting CAR in combination with a kinase inhibitor (e.g., one or more of a CDK4 / 6 inhibitor, a BTK inhibitor, an mTOR inhibitor, an MNK inhibitor, a dual PI3K / mTOR inhibitor, or a combination thereof). In some embodiments, the combination maintains or is better in clinical efficacy than either monotherapy. The present invention provides Further, the present invention relates to the use of cells, e.g., immune effector cells (e.g., T cells or NK cells), engineered to express a CAR molecule that binds a B cell antigen, e.g., CD19, in combination with a kinase inhibitor (e.g., one or more kinase inhibitors selected from a cyclin-dependent kinase 4 (CDK4) inhibitor, a Bruton's tyrosine kinase (BTK) inhibitor, an mTOR inhibitor, a mitogen-activated protein kinase-interacting kinase (MNK) inhibitor, a dual phosphatidylinositol 3-kinase (PI3K) / mTOR inhibitor, or a combination thereof), to treat a disorder associated with expression of a B cell antigen, e.g., CD19 (e.g., a cancer, e.g., a hematological cancer).

[0007]

[0013] Accordingly, in one aspect, the present invention relates to a method of treating a subject, e.g., a mammal, having a disease associated with expression of a B cell antigen, e.g., CD19. The method includes administering to the mammal an effective amount of a cell, e.g., an immune effector cell (e.g., a T cell or NK cell), expressing a CAR molecule that binds to the B cell antigen, in combination with a kinase inhibitor, e.g., a kinase inhibitor described herein. In some embodiments, the CAR molecule binds to CD19, e.g., a CAR molecule that binds to CD19 described herein. In other embodiments, the CAR molecule binds to one or more of CD20, CD22, or ROR1.

[0008] In some embodiments, the disease associated with expression of a B cell antigen (e.g., expression of one or more of CD19, CD20, CD22, or ROR1) is a proliferative disease such as a cancer, a malignant tumor, or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or a preleukemia, or a non-cancer-related disease associated with expression of one or more B cell antigens, e.g., CD19, CD20, CD22, or ROR1. In some embodiments, the disease is a solid or liquid tumor. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the disease is a blood cancer. In some embodiments, the blood cancer is leukemia. In some embodiments, the cancer is selected from the group consisting of one or more acute leukemias, including but not limited to B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), acute lymphoid leukemia (ALL); and one or more chronic leukemias, including but not limited to chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL). Additional blood cancers or hematological conditions include, but are not limited to, mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom's hypergammaglobulinemia. In certain embodiments, diseases associated with B cell antigen (e.g., one or more of CD19, CD20, CD22, or ROR1) expression are "preleukemias," a heterogeneous collection of hematological conditions united by the inadequate production (or dysplasia) of myeloid blood cells. In certain embodiments, diseases associated with B cell antigen (e.g., one or more of CD19, CD20, CD22, or ROR1) expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative disorders that express B cell antigens (e.g., one or more of CD19, CD20, CD22, or ROR1).Any combination of diseases associated with expression of the B cell antigens described herein (e.g., one or more of CD19, CD20, CD22, or ROR1) can be treated by the methods and compositions described herein.

[0009] In some embodiments, the disease associated with expression of a B cell antigen (e.g., one or more of CD19, CD20, CD22, or ROR1) is a lymphoma, e.g., MCL, Hodgkin's lymphoma, or DLBCL. In some embodiments, the disease associated with expression of a B cell antigen (e.g., one or more of CD19, CD20, CD22, or ROR1) is a leukemia, e.g., SLL, CLL, and / or ALL. In some embodiments, the disease associated with expression of a B cell antigen is multiple myeloma, e.g., multiple myeloma that is CD19-negative, e.g., as detected by both flow cytometry and RT-PCR, e.g., multiple myeloma in which the majority (99.95%) of neoplastic plasma cells have a CD19-negative phenotype.

[0010] In some embodiments, the kinase inhibitor is a CDK4 inhibitor, e.g., a CDK4 inhibitor described herein, e.g., a CD4 / 6 inhibitor, such as, e.g., 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one, hydrochloride (also referred to as palbociclib or PD0332991). In some embodiments, the kinase inhibitor is a BTK inhibitor, e.g., a BTK inhibitor described herein, such as, e.g., ibrutinib. In some embodiments, the kinase inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitor described herein, such as, e.g., rapamycin, a rapamycin analog, OSI-027. The mTOR inhibitor can be, e.g., an mTORCl inhibitor and / or an mTORC2 inhibitor, e.g., an mTORCl inhibitor and / or an mTORC2 inhibitor described herein. In some embodiments, the kinase inhibitor is an MNK inhibitor, e.g., an MNK inhibitor described herein, such as 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine. The MNK inhibitor can be, for example, an MNK1a, MNK1b, MNK2a, and / or MNK2b inhibitor. In some embodiments, the inhibitor can be a dual PI3K / mTOR inhibitor, e.g., PF-04695102.

[0011] In some embodiments, the kinase inhibitor is aloisine A; flavopiridol or HMR-1275, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone; crizotinib (PF-02341066); 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyra 1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine (RAF265); indisulam (E7070); roscovitine (CYC202); palbociclib (PD0332991); dinaciclib (SCH727965); N-[5-[[(5-te rt-Butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide (BMS387032); 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid (MLN8054); 5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridine The CDK4 inhibitor is selected from the group consisting of methanamine (AG-024322); 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid N-(piperidin-4-yl)amide (AT7519); 4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine (AZD5438); XL281 (BMS908662); and ribociclib.

[0012] In some embodiments, the kinase inhibitor is a DK4 inhibitor, e.g., palbociclib (PD0332991), and palbociclib is administered at a dose of about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g., 75 mg, 100 mg, or 125 mg) daily for a period of time, e.g., daily for 14 to 21 days of a 28-day cycle or daily for 7 to 12 days of a 21-day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more cycles of palbociclib are administered.

[0013] In some embodiments, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In preferred embodiments, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2-inducible kinase (ITK) and is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13.

[0014] In some embodiments, the kinase inhibitor is a BTK inhibitor, e.g., ibrutinib (PCI-32765), and ibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g., 250 mg, 420 mg, or 560 mg) daily for a period of time, e.g., daily in a 21-day cycle or daily in a 28-day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more cycles of ibrutinib are administered.

[0015] In some embodiments, the kinase inhibitor is temsirolimus; ridaforolimus (1R,2R,4S)-4-[(2R)-2-[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0 4,9 ]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate (also known as AP23573 and MK8669); everolimus (RAD001); rapamycin (AY22989); semapimod; (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502); and N 2 -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-, inner salt (SF1126); and XL765.

[0016] In some embodiments, the kinase inhibitor is an mTOR inhibitor, e.g., rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg) per day for a period of time, e.g., daily for a 21-day cycle or daily for a 28-day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of rapamycin are administered. In some embodiments, the kinase inhibitor is an mTOR inhibitor, e.g., everolimus, and the everolimus is administered at a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg) per day for a period of time, e.g., daily for a 28-day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of everolimus are administered.

[0017] In some embodiments, the kinase inhibitor is an MNK inhibitor selected from CGP052088; 4-amino-3-(p-fluorophenylamino)-pyrazolo[3,4-d]pyrimidine (CGP57380); cercosporamide; ETC-1780445-2; and 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine.

[0018] In some embodiments, the kinase inhibitor is 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF-04691502); N-[4-[[4-(dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N'-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]- phenyl]urea (PF-05212384, PKI-587); 2-methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile (BEZ-235); apitolisib (GDC-0980, RG7422); 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl yl}benzenesulfonamide (GSK2126458); 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-one maleic acid (NVP-BGT226); 3-[4-(4-morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol (PI-103); 5-(9 -isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (VS-5584, SB2343); and N-[2-[(3,5-dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide (XL765).

[0019] In one embodiment, the cells express a CAR molecule comprising an anti-CD19 binding domain (e.g., a murine or humanized antibody or antibody fragment that specifically binds to CD19), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a costimulatory domain and / or a primary signaling domain). In one embodiment, the CAR comprises an antibody or antibody fragment that comprises an anti-CD19 binding domain described herein (e.g., a murine or humanized antibody or antibody fragment that specifically binds to CD19 described herein), a transmembrane domain described herein, and an intracellular signaling domain described herein (e.g., an intracellular signaling domain described herein comprising a costimulatory domain and / or a primary signaling domain).

[0020] In some embodiments, the CAR molecule is capable of binding to CD19 (e.g., wild-type or mutant human CD19). In some embodiments, the CAR molecule comprises an anti-CD19 binding domain comprising one or more (e.g., all three) light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of an anti-CD19 binding domain described herein, and one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of an anti-CD19 binding domain described herein, e.g., an anti-CD19 binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of an anti-CD19 binding domain described herein, e.g., the anti-CD19 binding domain has two variable heavy chain regions, each comprising an HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the anti-CD19 binding domain comprises a murine light chain variable region described herein (e.g., in Table 7) and / or a murine heavy chain variable region described herein (e.g., in Table 7). In one embodiment, the anti-CD19 binding domain is an scFv comprising a murine light chain and a murine heavy chain of the amino acid sequences in Table 7. In one embodiment, the anti-CD19 binding domain (e.g., scFv) comprises a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided in Table 7, or a sequence with 95-99% identity to an amino acid sequence of Table 7; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided in Table 7, or a sequence with 95-99% identity to an amino acid sequence of Table 7. In one embodiment, the anti-CD19 binding domain comprises a sequence of SEQ ID NO: 59, or a sequence with 95-99% identity thereto.In one embodiment, the anti-CD19 binding domain is an scFv, wherein a light chain variable region comprising an amino acid sequence described herein, e.g., in Table 7, is linked to a heavy chain variable region comprising an amino acid sequence described herein, e.g., in Table 7, via a linker, e.g., a linker described herein. In one embodiment, the anti-CD19 binding domain comprises a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53). The light chain variable region and heavy chain variable region of the scFv can be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0021] In one embodiment, the CAR molecule comprises a humanized anti-CD19 binding domain comprising one or more (e.g., all three) light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of a humanized anti-CD19 binding domain described herein, and one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a humanized anti-CD19 binding domain described herein, e.g., an anti-CD19 binding domain comprising one or more, e.g., all three, humanized LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In some embodiments, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a humanized anti-CD19 binding domain described herein, e.g., the humanized anti-CD19 binding domain has two variable heavy chain regions, each comprising an HC CDR1, an HC CDR2, and an HC CDR3 described herein. In some embodiments, the humanized anti-CD19 binding domain comprises at least an HC CDR2. In some embodiments, the light chain variable region comprises one, two, three, or all four framework regions of the VK3_L25 germline sequence. In some embodiments, the light chain variable region comprises a modification (e.g., a substitution, e.g., a substitution of one or more amino acids found at the corresponding positions in the murine light chain variable region of SEQ ID NO: 58, e.g., a substitution at one or more positions 71 and 87). In some embodiments, the heavy chain variable region comprises one, two, three, or all four framework regions of the VH4_4-59 germline sequence. In some embodiments, the heavy chain variable region comprises a modification (e.g., a substitution, e.g., a substitution of one or more amino acids found at corresponding positions in the murine heavy chain variable region of SEQ ID NO: 58, e.g., one or more substitutions at positions 71, 73, and 78). In some embodiments, the humanized anti-CD19 binding domain comprises a light chain variable region described herein (e.g., in Table 3) and / or a heavy chain variable region described herein (e.g., in Table 3).In one embodiment, the humanized anti-CD19 binding domain is an scFv comprising a light chain and a heavy chain of the amino acid sequence in Table 3. In one embodiment, the humanized anti-CD19 binding domain (e.g., an scFv) comprises a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided in Table 3, or a sequence with 95-99% identity to an amino acid sequence of Table 3; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided in Table 3, or a sequence with 95-99% identity to an amino acid sequence of Table 3. In some embodiments, the humanized anti-CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, or a sequence having 95-99% identity thereto. In some embodiments, the humanized anti-CD19 binding domain is an scFv, in which a light chain variable region comprising an amino acid sequence as described herein, for example, in Table 3, is linked to a heavy chain variable region comprising an amino acid sequence as described herein, for example, in Table 3, via a linker, for example, a linker described herein. In some embodiments, the humanized anti-CD19 binding domain comprises a (Gly4-Ser)n linker (where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4) (SEQ ID NO:53). The light chain variable region and heavy chain variable region of the scFv may be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0022] In some embodiments, the CAR molecule comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 15. In some embodiments, the transmembrane domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 20, 10, or 5 modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 15, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 15.

[0023] In some embodiments, the anti-CD19 binding domain is connected to the transmembrane domain by a hinge region, e.g., a hinge region described herein. In some embodiments, the encoded hinge region comprises SEQ ID NO: 14 or SEQ ID NO: 45, or a sequence having 95-99% identity thereof.

[0024] In some embodiments, the CAR molecule further comprises a sequence encoding a costimulatory domain, e.g., a costimulatory domain described herein. In some embodiments, the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137). In some embodiments, the costimulatory domain comprises the sequence of SEQ ID NO: 16. In some embodiments, the costimulatory domain comprises the sequence of SEQ ID NO: 51. In some embodiments, the costimulatory domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 20, 10, or 5 modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51.

[0025] In some embodiments, the CAR molecule further comprises a sequence encoding an intracellular signaling domain, e.g., an intracellular signaling domain described herein. In some embodiments, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3 zeta. In some embodiments, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 and / or the sequence of SEQ ID NO: 17. In some embodiments, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 and / or the sequence of SEQ ID NO: 43. In some embodiments, the intracellular signaling domain comprises a functional signaling domain of CD27 and / or a functional signaling domain of CD3 zeta. In some embodiments, the intracellular signaling domain comprises the sequence of SEQ ID NO: 51 and / or the sequence of SEQ ID NO: 17. In some embodiments, the intracellular signaling domain comprises the sequence of SEQ ID NO: 51 and / or the sequence of SEQ ID NO: 43. In some embodiments, the intracellular signaling domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 20, 10, or 5 modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and / or the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and / or the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 43. In some embodiments, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 or SEQ ID NO: 51 and the sequence of SEQ ID NO: 17 or SEQ ID NO: 43, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

[0026] In some embodiments, the CAR molecule further comprises a leader sequence, e.g., a leader sequence described herein. In some embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 13, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 13.

[0027] In one embodiment, the CAR molecule comprises a leader sequence, e.g., a leader sequence described herein, e.g., a leader sequence having SEQ ID NO: 13 or having 95-99% identity thereof; an anti-CD19 binding domain described herein, e.g., an anti-CD19 binding domain comprising LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 described herein, e.g., a murine anti-CD19 binding domain described in Table 7, a humanized anti-CD19 binding domain described in Table 3, or a sequence with 95-99% identity thereof; a hinge region, e.g., a hinge region described herein, e.g., a hinge region having SEQ ID NO: 14 or having 95-99% identity thereof; a transmembrane domain, e.g., a transmembrane domain described herein, e.g., a transmembrane domain having a sequence of SEQ ID NO: 15 or a sequence with 95-99% identity thereof; an intracellular signaling domain, e.g., an intracellular signaling domain described herein (e.g., an intracellular signaling domain comprising a costimulatory domain and / or a primary signaling domain). In one embodiment, the intracellular signaling domain comprises a costimulatory domain, e.g., a costimulatory domain described herein, e.g., a 4-1BB costimulatory domain having a sequence of, or having 95-99% identity to, SEQ ID NO: 16 or SEQ ID NO: 51, and / or a primary signaling domain, e.g., a primary signaling domain described herein, e.g., a CD3 zeta stimulatory domain having a sequence of, or having 95-99% identity to, SEQ ID NO: 17 or SEQ ID NO: 43.

[0028] In some embodiments, the CAR molecule comprises the amino acid sequence of SEQ ID NO:58, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42, or at least one, two, or three of the amino acid sequences of SEQ ID NO:58, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42. , 3, 4, 5, 10, 15, 20 or 30 modifications (e.g., substitutions) but not more than 60, 50 or 40 modifications (e.g., substitutions) or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:58, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42.

[0029] In some embodiments, the cell expressing the CAR molecule comprises a vector comprising a nucleic acid sequence that expresses the CAR molecule. In some embodiments, the vector is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector further comprises a promoter. In some embodiments, the promoter is an EF-1 promoter. In some embodiments, the EF-1 promoter comprises the sequence of SEQ ID NO: 100. In some embodiments, the vector is an in vitro transcription vector, e.g., a vector that transcribes RNA of a nucleic acid molecule described herein. In some embodiments, the nucleic acid sequence in the in vitro vector further comprises a poly(A) tail, e.g., a polyA tail described herein, e.g., comprising about 150 adenosine bases (SEQ ID NO: 104). In some embodiments, the nucleic acid sequence in the in vitro vector further comprises a 3' UTR, e.g., comprising at least one repeat of a 3' UTR derived from human beta-globulin, e.g., a 3' UTR described herein. In some embodiments, the nucleic acid sequence in the in vitro vector further comprises a promoter, for example, a T2 promoter.

[0030] In certain embodiments of the compositions and methods disclosed herein, the cell expressing a CAR molecule (also referred to herein as a "CAR-expressing cell") is a cell or population of cells described herein, e.g., a human immune effector cell or population of cells (e.g., a human T cell or a human NK cell, e.g., a human T cell described herein or a human NK cell described herein). In certain embodiments, the human T cell is a CD8 +In some embodiments, the cell is a T cell. In some embodiments, the cell is an autologous T cell. In some embodiments, the cell is an allogeneic T cell. In some embodiments, the cell is a T cell, and the T cell is diacylglycerol kinase (DGK) deficient. In some embodiments, the cell is a T cell, and the T cell is Ikaros deficient. In some embodiments, the cell is a T cell, and the T cell is both DGK and Ikaros deficient. Compositions and methods disclosed herein referring to the term "cell" should be construed to encompass compositions and methods comprising one or more cells, e.g., a population of cells.

[0031] In other embodiments, cells expressing a CAR molecule, e.g., as described herein, can further express other agents, e.g., agents that enhance the activity of the CAR-expressing cells.

[0032] In some embodiments, the method further comprises administering cells expressing a CAR molecule as described herein in combination with an agent that enhances the activity of the CAR-expressing cells, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib. In some embodiments, the agent is a cytokine, e.g., IL-7, IL-15, IL-21, or a combination thereof. In some embodiments, the method comprises administering IL-7 to the subject. The cytokine can be delivered in combination with, e.g., simultaneously with, or shortly after, administration of the CAR-expressing cells. Alternatively, the cytokine can be delivered long term after administration of the CAR-expressing cells, e.g., after evaluation of the subject's response to the CAR-expressing cells.

[0033] In other embodiments, the agent that enhances the activity of a CAR-expressing cell can be an agent that inhibits an immune inhibitory molecule. Examples of immune inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In some embodiments, the agent that inhibits an immune inhibitory molecule comprises a first polypeptide, e.g., an immune inhibitory molecule, linked to a second polypeptide that transmits a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR beta, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described herein) and / or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein)). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a CD3 zeta signaling domain described herein).

[0034] In some embodiments, lymphocyte infusion, e.g., allogeneic lymphocyte infusion, is used to treat cancer, wherein the lymphocyte infusion comprises at least one CAR-expressing cell that binds a B-cell antigen (e.g., CD19), as described herein (also referred to herein as a CD19 CAR-expressing cell). In some embodiments, autologous lymphocyte infusion is used to treat cancer, wherein the autologous lymphocyte infusion comprises at least one CD19-expressing cell.

[0035] In one embodiment, the CD19 CAR-expressing cells, e.g., T cells, are administered to a subject who has previously undergone a stem cell transplant, e.g., an autologous stem cell transplant.

[0036] In some embodiments, CD19 CAR-expressing cells, e.g., T cells, are administered to a subject who has previously received melphalan.

[0037] In one embodiment, the cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered in combination with an agent that mitigates one or more side effects associated with administration of cells expressing a CAR molecule, e.g., an agent described herein.

[0038] In some embodiments, the kinase inhibitor is administered in combination with an agent that mitigates one or more side effects associated with administration of the kinase inhibitor, e.g., an agent described herein.

[0039] In one embodiment, the cells expressing a CAR molecule, e.g., a CAR molecule described herein, and the kinase inhibitor are administered in combination with an additional agent that treats a disease associated with CD19, e.g., an additional agent described herein.

[0040] In some embodiments, cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered at a dose and / or dosing schedule described herein.

[0041] In some embodiments, the CAR molecule is introduced into T cells, for example, using in vitro transcription, and the subject (e.g., human) receives an initial administration of cells comprising a CAR molecule and one or more subsequent administrations of cells comprising a CAR molecule, wherein the one or more subsequent administrations are administered within 15 days, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In some embodiments, the subject receives one or more administrations of cells comprising a CAR molecule per week, for example, two, three, or four administrations of cells comprising a CAR molecule per week. In some embodiments, the subject (e.g., human subject) receives one or more administrations of cells comprising a CAR molecule per week (e.g., two, three, or four administrations per week) (also referred to herein as a cycle), followed by a one-week break from the cells comprising a CAR molecule, and then receives one or more additional administrations of cells comprising a CAR molecule (e.g., one or more administrations per week of cells comprising a CAR molecule). In other embodiments, a subject (e.g., a human subject) receives more than one cycle of cells comprising a CAR molecule, with the interval between each cycle being less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In some embodiments, cells comprising a CAR molecule are administered every other day, three times a week. In some embodiments, cells comprising a CAR molecule are administered for at least 2, 3, 4, 5, 6, 7, 8, or more weeks.

[0042] In some embodiments, the combination of a kinase inhibitor and cells expressing a CAR molecule, e.g., a CAR molecule described herein, is administered as a first line treatment for a disease, e.g., cancer, e.g., a cancer described herein. In other embodiments, the combination of a kinase inhibitor and cells expressing a CAR molecule, e.g., a CAR molecule described herein, is administered as a second, third, or fourth line treatment for a disease, e.g., cancer, e.g., a cancer described herein.

[0043] In some embodiments, a cell (e.g., a population of cells) described herein is administered to a subject.

[0044] In some embodiments, the method involves administering a population of cells, a majority of which comprise a CAR molecule described herein. In some embodiments, the population of CAR-expressing cells comprises a mixture of cells expressing different CARs. For example, in some embodiments, the population of CAR-expressing cells can include a first cell expressing a CAR having an anti-CD19 binding domain described herein and a second cell expressing a CAR having a different anti-CD19 binding domain, e.g., an anti-CD19 binding domain described herein that is different from the anti-CD19 binding domain in the CAR expressed by the first cell. As another example, the population of CAR-expressing cells can include a first cell expressing a CAR comprising an anti-CD19 binding domain, e.g., as described herein, and a second cell expressing a CAR comprising an antigen binding domain against a target other than CD19 (e.g., CD123 or mesothelin). In some embodiments, the population of CAR-expressing cells includes, e.g., a first cell expressing a CAR comprising a primary intracellular signaling domain and a second cell expressing a CAR comprising a secondary signaling domain.

[0045] In some embodiments, the method includes administering a population of cells, wherein at least one cell in the population expresses a CAR having an anti-CD19 domain described herein and an agent that enhances the activity of the CAR-expressing cell, e.g., a second cell expresses an agent that enhances the activity of the CAR-expressing cell. For example, in some embodiments, the agent can be an agent that inhibits an immune inhibitory molecule. Examples of immune inhibitory molecules include PD1, PD-L1, CTLA-4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In some embodiments, the agent that inhibits an immune inhibitory molecule includes a first polypeptide, e.g., an inhibitory molecule, linked to a second polypeptide that transmits a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR beta, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described herein) and / or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein)). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a CD3 zeta signaling domain described herein).

[0046] In another aspect, the invention pertains to a cell expressing a CAR molecule described herein for use as a medicament in combination with a kinase inhibitor, e.g., a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib). In another aspect, the invention pertains to a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib) for use as a medicament in combination with a cell expressing a CAR molecule described herein.

[0047] In another aspect, the invention pertains to cells expressing a CAR molecule described herein for use in combination with a kinase inhibitor, e.g., a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib), in the treatment of a disease expressing a B cell antigen (e.g., CD19). In another aspect, the invention pertains to a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib), for use in combination with cells expressing a CAR molecule described herein, in the treatment of a disease expressing a B cell antigen (e.g., CD19). The disease can be, for example, a cancer, such as a hematological cancer. The cancer can be, for example, lymphoma, CLL, MCL, ALL, DLBCL, multiple myeloma, or other cancers described herein.

[0048] In another aspect, the invention pertains to a cell expressing a CAR molecule described herein for use as a medicament in combination with a cytokine, e.g., IL-7, IL-15 and / or IL-21 as described herein. In another aspect, the invention pertains to a cytokine described herein for use as a medicament in combination with a cell expressing a CAR molecule described herein.

[0049] In another aspect, the invention pertains to a cell expressing a CAR molecule described herein for use in combination with a cytokine, e.g., IL-7, IL-15 and / or IL-21 as described herein, in the treatment of a disease expressing CD 19. In another aspect, the invention pertains to a cytokine described herein for use in combination with a cell expressing a CAR molecule described herein, in the treatment of a disease expressing CD 19.

[0050] In another aspect, the invention features a method of treating a mammal having Hodgkin's lymphoma, comprising administering to the mammal an effective amount of a cell (e.g., a plurality of cells) expressing a CAR molecule, e.g., a CAR molecule described herein.

[0051] In one embodiment, the cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered in combination with an agent that increases the efficacy of the cells expressing the CAR molecule, e.g., an agent described herein.

[0052] In one embodiment, the cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered in combination with an agent that mitigates one or more side effects associated with administration of cells expressing a CAR molecule, e.g., an agent described herein.

[0053] In one embodiment, the cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered in combination with an agent treating Hodgkin's lymphoma, e.g., an agent described herein.

[0054] In some embodiments, cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered in combination with a low, immune-enhancing dose of an mTOR inhibitor, e.g., an mTOR inhibitor described herein. Without being bound by theory, it is believed that treatment with a low, immune-enhancing dose (e.g., a dose that is insufficient to completely suppress the immune system but sufficient to improve immune function) is accompanied by a decrease in PD-1-positive T cells or an increase in PD-1-negative cells. PD-1-positive T cells, but not PD-1-negative T cells, may be exhausted by binding to cells expressing a PD-1 ligand, e.g., PD-L1 or PD-L2.

[0055] In some embodiments, this approach can be used to optimize the performance of the CAR cells described herein in a subject. Without being bound by theory, it is believed that in some embodiments, the performance of endogenous, unmodified immune effector cells, e.g., T cells, is improved. Without being bound by theory, it is believed that in some embodiments, the performance of CD19 CAR-expressing cells is improved. In other embodiments, cells, e.g., T cells, that have been or will be engineered to express a CAR are treated ex vivo by contact with an mTOR inhibitor in an amount that increases the number of PD1-negative immune effector cells, e.g., T cells, or the ratio of PD1-negative immune effector cells, e.g., T cells / PD1-positive immune effector cells, e.g., T cells.

[0056] In some embodiments, administration of a low, immune-enhancing dose of an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or a catalytic inhibitor, is initiated prior to administration of a CAR-expressing cell, e.g., T cell, described herein. In some embodiments, the mTOR inhibitor is RAD001 or rapamycin. In some embodiments, the CAR cells are administered a sufficient time or in a sufficient amount after administration of the mTOR inhibitor such that the level of PD1-negative immune effector cells, e.g., T cells, or the ratio of PD1-negative immune effector cells, e.g., T cells / PD1-positive immune effector cells, e.g., T cells, is increased, at least transiently.

[0057] In some embodiments, the cells, e.g., immune effector cells (e.g., T cells or NK cells), to be engineered to express a CAR are harvested a sufficient time or amount after administration of a low, immune enhancing dose of an mTOR inhibitor such that the level of PD1 negative immune effector cells, e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g., T cells / PD1 positive immune effector cells, e.g., T cells, in the subject or harvested from the subject is increased, at least transiently.

[0058] In some embodiments, any of the methods described herein further include performing lymphodepletion on the subject, e.g., prior to administration of one or more cells expressing a CAR molecule described herein, e.g., a CAR molecule that binds CD19. Lymphodepletion can include, for example, administration of one or more of melphalan, cytoxan, cyclophosphamide, and fludarabine.

[0059] In some embodiments, the administered CAR-expressing cells comprise a regulatable CAR (RCAR), e.g., an RCAR described herein. The RCAR can comprise, e.g., an intracellular signaling member comprising an intracellular signaling domain and a first switch domain, an antigen binding member comprising an antigen binding domain that binds to CD19 and a second switch domain; and a transmembrane domain. The method can further comprise administering a dimerization molecule, e.g., in an amount sufficient to induce dimerization of the first switch domain and the second switch domain.

[0060] In some embodiments, the CAR-expressing cells and the kinase inhibitor are administered simultaneously or substantially simultaneously, e.g., as a first-line treatment. In some embodiments, a method administers a combination of a BTK inhibitor (e.g., ibrutinib) and a CAR-expressing cell (e.g., a CAR19-expressing cell) to a subject as a first-line treatment.

[0061] In other embodiments, the CAR-expressing cells and the kinase inhibitor are administered sequentially, for example, the kinase inhibitor is administered before the CAR-expressing cells or the CAR-expressing cells are administered before the kinase inhibitor.

[0062] In some embodiments, the disease associated with CD19 expression is a hematological cancer (e.g., a hematological cancer described herein, such as CLL, MCL, or ALL), and the subject is or is diagnosed as a partial responder, non-responder, or relapser to one or more therapies for the hematological cancer, e.g., a BTK inhibitor such as ibrutinib. In some embodiments, the subject has or is identified as having a BTK mutation. The mutation can be, for example, a point mutation, insertion, or deletion. The mutation can be, for example, a mutation at or near the binding site of the BTK inhibitor, e.g., the ATP-binding pocket. The mutation can contribute to a decreased response (e.g., resistance) to the BTK inhibitor.

[0063] In certain embodiments of any of the methods disclosed herein, the method includes administering a BTK inhibitor (e.g., ibrutinib) to a subject, reducing the amount of the BTK inhibitor (e.g., ceasing administration), and subsequently administering a CAR-expressing cell (e.g., a CAR19-expressing cell) to the subject.

[0064] In some embodiments, the method includes administering a BTK inhibitor (e.g., ibrutinib) to the subject, and subsequently administering a combination of the BTK inhibitor and a CAR-expressing cell (e.g., a CAR19-expressing cell) to the subject.

[0065] In some embodiments, the method includes administering a BTK inhibitor (e.g., ibrutinib) to a subject, reducing the amount of the BTK inhibitor (e.g., ceasing or discontinuing administration), and subsequently administering a combination of a CAR-expressing cell (e.g., a CAR19-expressing cell) and a second BTK inhibitor (e.g., a BTK inhibitor other than the first BTK inhibitor, e.g., other than ibrutinib) to the subject. In some embodiments, the second BTK inhibitor is selected from one or more of GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13, or a combination thereof.

[0066] In some embodiments, the disease associated with expression of a B-cell antigen (e.g., CD19) is a hematological cancer (e.g., a hematological cancer described herein, e.g., CLL, MCL, or ALL), and the method delays the onset of resistance or reduces resistance in the subject to a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), a CAR-expressing cell (e.g., a CAR19-expressing cell), or both. In some embodiments, the disease associated with expression of CD19 is a hematological cancer (e.g., a hematological cancer described herein, e.g., CLL, MCL, or ALL), and the method prolongs remission or delays relapse of the hematological cancer. For example, remission can be prolonged, relapse can be delayed, resistance onset can be delayed, or resistance can be reduced, compared to the expected course of the disease when treated with a monotherapy of the kinase inhibitor or the CAR-expressing cell.

[0067] Representative treatment regimens that can be used in any of the above methods include one or more of the following:

[0068] In some embodiments, the kinase inhibitor and a CAR-expressing cell (e.g., a CAR19-expressing cell) are administered to a subject, e.g., a mammal, as a first line of treatment.

[0069] In other embodiments, the CAR-expressing cells (e.g., CAR19-expressing cells) are administered to the subject, e.g., mammal, after administration of the kinase inhibitor.

[0070] In other embodiments, the CAR-expressing cells (e.g., CAR19-expressing cells) are administered after cessation of administration of the kinase inhibitor.

[0071] In other embodiments, administration of the kinase inhibitor is initiated prior to administration of the CAR19-expressing cells, and the CAR19-expressing cells are administered in combination with the ongoing administration of the kinase inhibitor.

[0072] In some embodiments, a subject is administered a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), e.g., as a first-line treatment. After a predetermined period of administration (e.g., 1 or 2 months, but also 2 weeks, 3 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 6 months, 9 months, 12 months, 15 months, or 18 months), a CAR-expressing cell (e.g., a CAR19-expressing cell) is administered to the subject, alone or in combination with the kinase inhibitor. In some embodiments, the subject's response to the treatment is evaluated at predetermined intervals, e.g., before or during treatment with the kinase inhibitor and / or CAR-expressing cell. If the evaluation indicates that the subject is a complete responder, the CAR-expressing cell (e.g., a CAR19-expressing cell) is not administered. If the evaluation indicates that the subject is a partial responder or has stable disease in response to the kinase inhibitor, the CAR-expressing cell (e.g., a CAR19-expressing cell) is administered in combination with the kinase inhibitor, e.g., as described herein. If the assessment indicates that the subject is a non-responder or a relapser, the CAR-expressing cells (e.g., CAR19-expressing cells) are administered in combination with a kinase inhibitor or a second kinase inhibitor, e.g., a second kinase inhibitor described herein.

[0073] In other embodiments, the subject, e.g., mammal, is or is identified as being a complete or partial responder to a BTK inhibitor (e.g., ibrutinib) or a complete or partial responder to CAR19-expressing cells.

[0074] In some embodiments, when a subject is (or is identified as) a complete responder to a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the subject is not administered CAR-expressing cells (e.g., CAR19-expressing cells) for the duration of the complete response. In other embodiments, when a subject is (or is identified as) a complete responder to a kinase inhibitor (e.g., a complete responder to ibrutinib), the subject is administered CAR-expressing cells (e.g., CAR19-expressing cells) for the duration of the complete response. In some embodiments, after administration of CAR-expressing cells (e.g., CAR19-expressing cells), the subject experiences a prolonged response or delayed relapse (e.g., compared to the expected course of the disease when treated without CAR therapy).

[0075] In some embodiments, when a subject is (or is identified as) a partial responder to a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the subject is not administered CAR-expressing cells (e.g., CAR19-expressing cells) during the period of partial response. In other embodiments, when a subject is (or is identified as) a partial responder to a kinase inhibitor, the subject is administered CAR-expressing cells (e.g., CAR19-expressing cells) (alone or in combination with a BTK inhibitor) during the period of partial response. In some embodiments, after CAR therapy, the subject experiences a complete response and / or a prolonged response or delayed relapse (e.g., compared to the expected course of the disease when treated without CAR therapy).

[0076] In some embodiments, when a subject has (or is identified as having) stable disease after treatment with a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the subject is not administered CAR therapy during the period of stable disease. In other embodiments, when a subject has (or is identified as having) stable disease after treatment with a kinase inhibitor, the subject is administered CAR therapy during the period of stable disease. In some embodiments, after CAR therapy, the subject experiences a partial response, a complete response, and / or a prolonged response or delayed relapse (e.g., compared to the expected course of disease when treated without CAR therapy).

[0077] In some embodiments, when a subject has (or is identified as having) progressive disease after treatment with a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the subject is not administered a CAR-expressing cell (e.g., a CAR19-expressing cell) during the period of progressive disease. In other embodiments, when a subject has (or is identified as having) progressive disease after treatment with a kinase inhibitor, the subject is administered a CAR-expressing cell (e.g., a CAR19-expressing cell) during the period of progressive disease. In some embodiments, after CAR therapy, the subject experiences stable disease, a partial response, a complete response, and / or a prolonged response or delayed relapse (e.g., compared to the expected course of disease when treated without CAR therapy).

[0078] In other embodiments, when the mammal is or is identified as a non-responder or relapser to ibrutinib, the CAR-expressing cells are administered in combination with a second kinase inhibitor, wherein the second kinase inhibitor is other than ibrutinib. The second kinase inhibitor can be one or more selected from GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13, or a combination thereof.

[0079] In other embodiments, the subject, e.g., mammal, is (or is identified as) a partial responder to a kinase inhibitor, and the subject is administered a CAR-expressing cell (e.g., a CAR19-expressing cell), alone or in combination with a BTK inhibitor, for the duration of the partial response.

[0080] In other embodiments, the subject, e.g., mammal, is (or is identified as) a non-responder who has progressive or stable disease after treatment with ibrutinib, and the subject is administered a CAR-expressing cell (e.g., a CAR19-expressing cell) in combination with a second BTK inhibitor during the period of progressive or stable disease, where the second kinase inhibitor is other than ibrutinib.

[0081] In another aspect, a method of treating a subject, e.g., a mammal, having a disease associated with expression of a B cell antigen (e.g., CD19) is provided. The method comprises administering to the subject effective amounts of a kinase inhibitor described herein (e.g., a BTK kinase inhibitor described herein, e.g., ibrutinib) and a CAR-expressing cell (e.g., a CAR19-expressing cell) in combination (e.g., simultaneously (or substantially simultaneously) or sequentially).

[0082] In some embodiments, the kinase inhibitor and the CAR-expressing cells (e.g., CAR19 cells) are administered in combination, e.g., as a first line of treatment.

[0083] In some embodiments, the kinase inhibitor is administered initially, e.g., as monotherapy or first-line treatment; the amount of the kinase inhibitor is reduced (e.g., discontinued or interrupted), and then a CAR-expressing cell (e.g., a CAR19-expressing cell) is administered to the subject.

[0084] In other embodiments, the kinase inhibitor is administered first, e.g., as monotherapy or first-line treatment; subsequently, a combination of the kinase inhibitor and a CAR-expressing cell (e.g., a CAR19-expressing cell) is administered to the subject.

[0085] In other embodiments, the kinase inhibitor is administered initially, e.g., as monotherapy or first-line treatment; after the amount of the kinase inhibitor is reduced (e.g., discontinued or interrupted), a combination of a second kinase inhibitor and a CAR-expressing cell (e.g., a CAR19-expressing cell) is administered to the subject.

[0086] In some embodiments, the subject's response to treatment is assessed at scheduled intervals, for example, before or during treatment with a kinase inhibitor and / or CAR-expressing cells. If the assessment shows that the subject is a complete responder, CAR-expressing cells (e.g., CAR19-expressing cells) are not administered. If the assessment shows that the subject is a partial responder or has stable disease in response to the kinase inhibitor, CAR-expressing cells (e.g., CAR19-expressing cells) are administered in combination with a kinase inhibitor, for example, as described herein. If the assessment shows that the subject is a non-responder or relapser, CAR-expressing cells (e.g., CAR19-expressing cells) are administered in combination with a kinase inhibitor or a second kinase inhibitor, for example, a second kinase inhibitor described herein.

[0087] In some embodiments, the disease associated with expression of a B cell antigen (e.g., CD19) is a hematological cancer, leukemia, lymphoma, MCL, CLL, ALL, Hodgkin's lymphoma, or multiple myeloma.

[0088] In some embodiments, the kinase inhibitor is a BTK inhibitor selected from ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13; a CDK4 inhibitor selected from palbociclib, aloisine A, flavopiridol, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone; crizotinib (PF-02341066, P276-00, RAF265, indisulam, roscovitine, dinaciclib, an mTOR inhibitor selected from BMS387032, MLN8054, AG-024322, AT7519, AZD5438, BMS908662; or ribociclib; rapamycin, or a rapamycin analog such as everolimus, temsirolimus, ridaforolimus, semapimod, AZD8055, PF04691502, SF1126, XL765, or OSI-027; or an MNK inhibitor selected from CGP052088, CGP57380, cercosporamide, or ETC-1780445-2, or a 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine.

[0089] In one aspect, the invention relates to a method of treating or providing anti-tumor immunity to a subject, e.g., a mammal, with Hodgkin's lymphoma. The method comprises administering to the subject an effective amount of cells expressing a CAR molecule that binds CD19, alone or in combination with a second therapy.

[0090] In another aspect, the present invention relates to a method of treating or providing anti-tumor immunity to a subject, e.g., a mammal, with multiple myeloma (e.g., CD19-positive multiple myeloma or CD19-negative myeloma). In one embodiment, the multiple myeloma is CD19-negative, e.g., a majority (99.95%) of neoplastic plasma cells have a CD19-negative phenotype, e.g., as detected by both flow cytometry and RT-PCR. The method comprises administering to the subject an effective amount of cells expressing a CAR molecule that binds CD19, alone or in combination with a second therapy (e.g., standard of care for multiple myeloma). The method may further comprise administering a kinase inhibitor as described herein.

[0091] In some embodiments of methods related to Hodgkin's lymphoma or multiple myeloma, the CAR molecule is a humanized CAR molecule, e.g., as described herein. In some embodiments, the CAR molecule is a CAR molecule described herein. For example, in some embodiments, the CAR molecule comprises an anti-CD19 binding domain that includes one or more (e.g., two, three, four, five, or all) of: an LC CDR1 of SEQ ID NO: 5, an LC CDR2 of SEQ ID NO: 26, and an LC CDR3 of SEQ ID NO: 27; an HC CDR1 of SEQ ID NO: 19, an LC CDR2 of any of SEQ ID NOs: 20-23, and an HC CDR3 of SEQ ID NO: 24.

[0092] In some embodiments of the methods relating to Hodgkin's lymphoma or multiple myeloma, a CAR molecule (e.g., CART19 or CTL019) is administered as monotherapy. In some embodiments, the method further comprises administering a kinase inhibitor, e.g., a BTK inhibitor (e.g., ibrutinib), a CDK4 inhibitor, an mTOR inhibitor, or an MNK inhibitor.

[0093] In some embodiments of the methods related to multiple myeloma, the CAR molecule (e.g., CART19 or CTL019) is administered in combination with a standard of care treatment for multiple myeloma, e.g., myeloablative chemotherapy and / or autologous stem cell transplant rescue (e.g., following melphalan administration (e.g., high-dose melphalan)).

[0094] In another aspect, the invention features a composition comprising cells expressing a CAR molecule that binds a B cell antigen (e.g., one or more of CD19, CD20, CD22, or ROR1) and one or more kinase inhibitors, wherein the kinase inhibitor is chosen from a Bruton's tyrosine kinase (BTK) inhibitor, a cyclin-dependent kinase 4 (CDK4) inhibitor, an mTOR inhibitor, or a mitogen-activated protein kinase-interacting kinase (MNK) inhibitor. The CAR-expressing cells and the one or more kinase inhibitors can be present in a single dosage form or in two or more dosage forms.

[0095] In some embodiments, the compositions disclosed herein are for use as pharmaceuticals.

[0096] In some embodiments, the compositions disclosed herein are used to treat diseases associated with expression of a B cell antigen (e.g., CD19).

[0097] Methods and compositions for producing CAR-expressing cells The present invention also provides, in certain aspects, a method of producing a population of immune effector cells (e.g., T cells or NK cells) that can be engineered to express a CAR (e.g., a CAR described herein), the method comprising: providing a population of immune effector cells; and contacting the immune effector cells with a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) under conditions sufficient to inhibit a target of the kinase inhibitor (e.g., BTK and / or ITK). The method further comprises contacting, e.g., transducing, the immune effector cells with a nucleic acid encoding the CAR molecule.

[0098] In one aspect, the invention provides a method of producing a CAR-expressing cell (e.g., a CAR-expressing immune effector cell or population of cells), comprising contacting the cell or population of cells with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib; and introducing (e.g., transducing) a nucleic acid encoding a CAR molecule into the cell or population of cells under conditions such that the CAR molecule is expressed.

[0099] In some embodiments of the methods of producing a CAR-expressing cell, the CAR molecule encoded by the nucleic acid is a CAR molecule that binds CD19. In some embodiments, the method further comprises culturing the one or more cells under conditions that allow the cells, or at least a subpopulation of the cells, to express the CAR molecule. In some embodiments, the cells are T cells or NK cells, or the population of cells includes T cells, NK cells, or both. In some embodiments, the method comprises contacting the one or more cells with a kinase inhibitor (e.g., for 10-20 minutes, 20-30 minutes, 30-40 minutes, 40-60 minutes, or 60-120 minutes), followed by removing most or all of the kinase inhibitor from the one or more cells. In some embodiments, the kinase inhibitor is added after harvesting the one or more cells or prior to stimulating the one or more cells. In some embodiments, the kinase inhibitor is a BTK inhibitor, a CDK4 inhibitor, an mTOR inhibitor, or an MNK inhibitor. In some embodiments, the kinase inhibitor is ibrutinib. In some embodiments, the population of cells also includes cancer cells, e.g., leukemia or lymphoma cells. The cancer cells may be, for example, CLL cells, MCL cells, or ALL cells. In some embodiments, the kinase inhibitor inhibits a target (e.g., BTK) in cancer cells, e.g., reduces its activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the kinase inhibitor inhibits a target (e.g., ITK) in immune effector cells, e.g., reduces its activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.

[0100] In some aspects, the present invention also provides a reaction mixture comprising a kinase inhibitor (e.g., a BTK inhibitor) and a CAR molecule or a nucleic acid encoding a CAR molecule. In some embodiments, the reaction mixture further comprises a population of immune effector cells.

[0101] In some embodiments, one or more of the immune effector cells express a CAR molecule or contain a nucleic acid encoding a CAR molecule. In some embodiments, the kinase inhibitor is selected from a BTK inhibitor, a CDK4 inhibitor, an mTOR inhibitor, or an MNK inhibitor. In some embodiments, the BTK inhibitor is selected from ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13. In some embodiments, the reaction mixture includes cancer cells, e.g., blood cancer cells. The cancer cells can be, for example, cells recovered from a subject when the immune effector cells are recovered from the subject.

[0102] In one aspect, the present invention provides a reaction mixture comprising a population of immune effector cells and a CAR molecule or a nucleic acid encoding a CAR molecule, wherein the immune effector cells comprise covalently inactivated ITK. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the ITK is covalently inactivated. In some embodiments, the reaction mixture further comprises cancer cells. In some embodiments, the cancer cells comprise covalently inactivated BTK. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the BTK is covalently inactivated. In some embodiments, BTK or ITK forms a covalent bond to a small molecule, such as ibrutinib, at or near its ATP-binding domain. In some embodiments, BTK forms a covalent bond to a small molecule, such as ibrutinib, at or near its cysteine-481.

[0103] In some embodiments, the reaction mixture as described herein further comprises a buffer or other reagent, e.g., a PBS-containing solution. In some embodiments, the reaction mixture further comprises an agent that activates and / or expands the cell population, e.g., an agent that stimulates a CD3 / TCR complex-associated signal and / or a ligand that stimulates a costimulatory molecule on the surface of the cell. In some embodiments, the agent is a bead conjugated with an anti-CD3 antibody or fragment thereof and / or an anti-CD28 antibody or fragment thereof. In some embodiments, the reaction mixture further comprises one or more factors for proliferation and / or viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additive for cell growth. In some embodiments, the reaction mixture further comprises IL-15 and / or IL-7. In some embodiments, the population of cells in the reaction mixture comprises a nucleic acid molecule, e.g., a nucleic acid molecule described herein, comprising a CAR-encoding sequence, e.g., a CD19 CAR-encoding sequence, e.g., as described herein. In some embodiments, the population of cells in the reaction mixture comprises a vector comprising a nucleic acid sequence encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein. In some embodiments, the vector is a vector described herein, e.g., a vector selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the reaction mixture further comprises a cryoprotectant or stabilizer, such as, for example, a sugar, an oligosaccharide, a polysaccharide, and a polyol (e.g., trehalose, mannitol, sorbitol, lactose, sucrose, glucose, and dextran), a salt, and a crown ether. In some embodiments, the cryoprotectant is dextran.

[0104] In some embodiments, the methods disclosed herein further comprise contacting the population of immune effector cells with a nucleic acid encoding a telomerase subunit, e.g., hTERT. The nucleic acid encoding the telomerase subunit can be DNA.

[0105] In some embodiments, the methods of manufacture disclosed herein further comprise culturing the population of immune effector cells in serum comprising 2% hAB serum.

[0106] Headings, subheadings, or numbered or lettered elements, e.g., (a), (b), (i), etc., are presented solely for ease of reading. The use of headings or numbered or lettered elements herein does not require that the steps or elements be performed in alphabetical order or that the steps or elements be necessarily separated from one another.

[0107] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0108] Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0109] [Figure 1-1] Figures 1A, 1B, and 1C are graphical representations of cytotoxicity assayed on ND317 (normal donor) T cells transduced with a murine anti-CD19 CAR or a humanized anti-CD19 CAR of the invention and cultured with control K562 cells that do not express CD19 (K562cc) as shown in Figure 1A, K562 cells transformed with CD19 (K562.CD19) as shown in Figure 1B, or malignant B cells isolated from a CLL patient (Pt14 B cell isolate) as shown in Figure 1C. [Figure 1-2]Figures 1A, 1B, and 1C are graphical representations of cytotoxicity assayed on ND317 (normal donor) T cells transduced with a murine anti-CD19 CAR or a humanized anti-CD19 CAR of the invention and cultured with control K562 cells that do not express CD19 (K562cc) as shown in Figure 1A, K562 cells transformed with CD19 (K562.CD19) as shown in Figure 1B, or malignant B cells isolated from a CLL patient (Pt14 B cell isolate) as shown in Figure 1C.

[0110] [Figure 2] Figures 2A and 2B are graphs showing the proliferative responses of humanized and murine anti-CD19 CAR-expressing cells to CD19+ cells, where high viable CAR+ T cell numbers correspond to populations showing maximal CD4+ and CD8+ T cell proliferation to primary CLL cells.

[0111] [Figure 3] FIG. 3 is a graphical representation of the deconvoluted HPLC mass spectra of scFvs of the invention, where the top row depicts the intact scFv and the bottom row depicts the cognate deglycosylated scFv.

[0112] [Figure 4] Figure 4 is a graphical representation of conformational stability measured by differential scanning fluorimetry. The Tm of the murine scFv was 57°C (bold line). The fully humanized scFv variants exhibited a higher Tm of approximately 70°C compared to the parental murine scFv. Residues introduced by humanization improved Tm by more than 10°C.

[0113] [Figure 5]Figure 5 is a graphical representation of CD19 CAR-transduced T cell proliferation, where CART19 cells are directed against (a) a chronic myeloid leukemia ("CML") cell line that is negative for expression of CD19 and therefore used as a negative control; (b) recombinant K562 cells that are positive for expression of CD19 and therefore used as a positive control; or (c) Pt14 B cells isolated from a CLL patient and expressing CD19 on their cell surface.

[0114] [Figure 6] 6A and 6B are schematic diagrams of representative CARs.

[0115] [Figure 7] Figure 7 describes HALLX5447 primary ALL disease progression after CD19-transduced CAR T cell treatment in NSG mice. Primary human ALL cells in NSG mice after treatment with CD19-specific CAR T cells showed control of disease progression. The mean percentage of CD19+ human ALL cells is an indicator of peripheral blood disease burden in NSG mice up to 65 days after tumor implantation. Black circles: mice treated with 100 μl of PBS via tail vein; red squares: mice treated with mock-transduced T cells; blue triangles: mice treated with murine CD19 CAR-transduced T cells; and purple inverted triangles: mice treated with humanized CD19 CAR-transduced T cells. Significance calculated by ANOVA; * denotes P<0.01.

[0116] [Figure 8] Figure 8 shows CD19 expression on tumor cells from a patient. CD138+ CD45dim tumor cells were stained for CD19 (x-axis) and CD38 (y-axis). Approximately 1-2% of tumor cells expressed the CD19 antigen.

[0117] [Figure 9] FIG. 9 is two graphs showing cell proliferation and cell size of CART19 cells upon treatment with increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM).

[0118] [Figure 10A-1] Figures 10A and 10B show the proliferation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. Figure 10A is a series of histograms showing the proliferation of CART19 cells stimulated with tumor cell lines MOLM14, JEKO-1, and RL in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). Cells were stained with CFSE and analyzed by flow cytometry to determine the percentage of proliferating cells, as indicated by the bars in each histogram. Figure 10B is a quantification of the representative histogram in Figure 10A. [Figure 10A-2] Figures 10A and 10B show the proliferation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. Figure 10A is a series of histograms showing the proliferation of CART19 cells stimulated with tumor cell lines MOLM14, JEKO-1, and RL in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). Cells were stained with CFSE and analyzed by flow cytometry to determine the percentage of proliferating cells, as indicated by the bars in each histogram. Figure 10B is a quantification of the representative histogram in Figure 10A. [Figure 10A-3] Figures 10A and 10B show the proliferation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. Figure 10A is a series of histograms showing the proliferation of CART19 cells stimulated with tumor cell lines MOLM14, JEKO-1, and RL in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). Cells were stained with CFSE and analyzed by flow cytometry to determine the percentage of proliferating cells, as indicated by the bars in each histogram. Figure 10B is a quantification of the representative histogram in Figure 10A. [Figure 10A-4]Figures 10A and 10B show the proliferation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. Figure 10A is a series of histograms showing the proliferation of CART19 cells stimulated with tumor cell lines MOLM14, JEKO-1, and RL in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). Cells were stained with CFSE and analyzed by flow cytometry to determine the percentage of proliferating cells, as indicated by the bars in each histogram. Figure 10B is a quantification of the representative histogram in Figure 10A. [Figure 10B] Figures 10A and 10B show the proliferation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. Figure 10A is a series of histograms showing the proliferation of CART19 cells stimulated with tumor cell lines MOLM14, JEKO-1, and RL in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). Cells were stained with CFSE and analyzed by flow cytometry to determine the percentage of proliferating cells, as indicated by the bars in each histogram. Figure 10B is a quantification of the representative histogram in Figure 10A.

[0119] [Figure 11A-1] Figures 11A and 11B show CD107a degranulation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. Figure 11A is a series of flow cytometry profiles showing CD107a degranulation of CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). CD107a expression is measured on the y-axis. Figure 11B is a quantification of the results from Figure 11A. [Figure 11A-2]Figures 11A and 11B show CD107a degranulation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. Figure 11A is a series of flow cytometry profiles showing CD107a degranulation of CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). CD107a expression is measured on the y-axis. Figure 11B is a quantification of the results from Figure 11A. [Figure 11A-3] Figures 11A and 11B show CD107a degranulation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. Figure 11A is a series of flow cytometry profiles showing CD107a degranulation of CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). CD107a expression is measured on the y-axis. Figure 11B is a quantification of the results from Figure 11A. [Figure 11A-4] Figures 11A and 11B show CD107a degranulation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. Figure 11A is a series of flow cytometry profiles showing CD107a degranulation of CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). CD107a expression is measured on the y-axis. Figure 11B is a quantification of the results from Figure 11A. [Figure 11A-5] Figures 11A and 11B show CD107a degranulation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. Figure 11A is a series of flow cytometry profiles showing CD107a degranulation of CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). CD107a expression is measured on the y-axis. Figure 11B is a quantification of the results from Figure 11A. [Figure 11A-6] Figures 11A and 11B show CD107a degranulation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. Figure 11A is a series of flow cytometry profiles showing CD107a degranulation of CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). CD107a expression is measured on the y-axis. Figure 11B is a quantification of the results from Figure 11A. [Figure 11B] Figures 11A and 11B show CD107a degranulation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. Figure 11A is a series of flow cytometry profiles showing CD107a degranulation of CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). CD107a expression is measured on the y-axis. Figure 11B is a quantification of the results from Figure 11A.

[0120] [Figure 12-1] Figure 12 is a series of flow cytometry profiles showing cytoplasmic IL-2 production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IL-2 expression. [Figure 12-2] Figure 12 is a series of flow cytometry profiles showing cytoplasmic IL-2 production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IL-2 expression. [Figure 12-3]Figure 12 is a series of flow cytometry profiles showing cytoplasmic IL-2 production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IL-2 expression. [Figure 12-4] Figure 12 is a series of flow cytometry profiles showing cytoplasmic IL-2 production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IL-2 expression. [Figure 12-5] Figure 12 is a series of flow cytometry profiles showing cytoplasmic IL-2 production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IL-2 expression. [Figure 12-6] Figure 12 is a series of flow cytometry profiles showing cytoplasmic IL-2 production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IL-2 expression.

[0121] [Figure 13-1] Figure 13 is a series of flow cytometry profiles showing cytoplasmic TNF-α production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents TNF-α expression. [Figure 13-2]Figure 13 is a series of flow cytometry profiles showing cytoplasmic TNF-α production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents TNF-α expression. [Figure 13-3] Figure 13 is a series of flow cytometry profiles showing cytoplasmic TNF-α production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents TNF-α expression. [Figure 13-4] Figure 13 is a series of flow cytometry profiles showing cytoplasmic TNF-α production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents TNF-α expression. [Figure 13-5] Figure 13 is a series of flow cytometry profiles showing cytoplasmic TNF-α production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents TNF-α expression. [Figure 13-6] Figure 13 is a series of flow cytometry profiles showing cytoplasmic TNF-α production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents TNF-α expression.

[0122] [Figure 14-1]Figure 14 is a series of flow cytometry profiles showing cytoplasmic IFN-g production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IFN-g expression. [Figure 14-2] Figure 14 is a series of flow cytometry profiles showing cytoplasmic IFN-g production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IFN-g expression. [Figure 14-3] Figure 14 is a series of flow cytometry profiles showing cytoplasmic IFN-g production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IFN-g expression. [Figure 14-4] Figure 14 is a series of flow cytometry profiles showing cytoplasmic IFN-g production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IFN-g expression. [Figure 14-5] Figure 14 is a series of flow cytometry profiles showing cytoplasmic IFN-g production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IFN-g expression. [Figure 14-6] Figure 14 is a series of flow cytometry profiles showing cytoplasmic IFN-g production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IFN-g expression.

[0123] [Figure 15-1] FIG. 15 is a series of graphs showing cytokine secretion from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). [Figure 15-2] FIG. 15 is a series of graphs showing cytokine secretion from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). [Figure 15-3] FIG. 15 is a series of graphs showing cytokine secretion from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). [Figure 15-4] FIG. 15 is a series of graphs showing cytokine secretion from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). [Figure 15-5] FIG. 15 is a series of graphs showing cytokine secretion from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). [Figure 15-6] FIG. 15 is a series of graphs showing cytokine secretion from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). [Figure 15-7] FIG. 15 is a series of graphs showing cytokine secretion from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). [Figure 15-8] FIG. 15 is a series of graphs showing cytokine secretion from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). [Figure 15-9] FIG. 15 is a series of graphs showing cytokine secretion from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). [Figure 15-10] FIG. 15 is a series of graphs showing cytokine secretion from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM).

[0124] [Figure 16-1] Figures 16A, 16B, 16C, 16D, 16E, and 16F are graphs showing the killing of CART19 tumor cells, MOLM14 (Figures 16A and 16D), JEKO (Figures 16B and 16E), and RL (Figures 16C and 16F), alone or in the presence of increasing concentrations of ibrutinib. Untreated (UTD) or CART19 cells were incubated with tumor cells at various ratios, and the total cell flux (Figures 16A, 16B, and 16C) and percentage of dead cells were assessed (16D, 16E, and 16F). [Figure 16-2] Figures 16A, 16B, 16C, 16D, 16E, and 16F are graphs showing the killing of CART19 tumor cells, MOLM14 (Figures 16A and 16D), JEKO (Figures 16B and 16E), and RL (Figures 16C and 16F), alone or in the presence of increasing concentrations of ibrutinib. Untreated (UTD) or CART19 cells were incubated with tumor cells at various ratios, and the total cell flux (Figures 16A, 16B, and 16C) and percentage of dead cells were assessed (16D, 16E, and 16F).

[0125] [Figure 17]Figures 17A, 17B, and 17C are graphic representations of tumor cell killing by CART19 after 24 hours, as measured by flow cytometry to count total cell number. Tumor cell lines MOLM14 (Figure 17A), JEKO (Figure 17B), and RL (Figure 17C) were incubated with untreated (UTD) or CART19 cells alone (alone) or in combination with various concentrations of ibrutinib.

[0126] [Figure 18-1] Figures 18A, 18B, 18C, and 18D are graphical representations of CART19 dose-finding in a RL MCL mouse model. Tumor burden was monitored over time by bioluminescence imaging (BLI) (Figures 18A and 18B). Overall survival was monitored over time (Figure 18C). [Figure 18-2] Figures 18A, 18B, 18C, and 18D are graphical representations of CART19 dose-finding in a RL MCL mouse model. Tumor burden was monitored over time by bioluminescence imaging (BLI) (Figures 18A and 18B). Overall survival was monitored over time (Figure 18C).

[0127] [Figure 19A] Figures 19A and 19B are graphical representations of CART19 dose finding in the JEKO-1 MCL mouse model. Tumor size was monitored over time by bioluminescence imaging (BLI) (Figure 19A), and overall survival was also monitored over time (Figure 19B). [Figure 19B] Figures 19A and 19B are graphical representations of CART19 dose finding in the JEKO-1 MCL mouse model. Tumor size was monitored over time by bioluminescence imaging (BLI) (Figure 19A), and overall survival was also monitored over time (Figure 19B).

[0128] [Figure 20] FIG. 20 is a schematic diagram showing the protocol for administering and evaluating CART19 and ibrutinib combination therapy in an in vivo mouse model.

[0129] [Figure 21] Figures 21A, 21B, 21C, and 21D are graphical representations showing the transduction efficiency of PBMCs to produce CART19 T cells when the cells were treated with ibrutinib before transduction. Untreated PBMCs were evaluated for CAR19 expression before (Figure 21A) and after (Figure 21C) transduction. Ibrutinib-treated PBMCs were evaluated for CAR19 expression before (Figure 21B) and after (Figure 21D) transduction.

[0130] [Figure 22] Figures 22A, 22B, 22C, 22D, and 22E are graphical representations of the effect of ibrutinib treatment on CD3 / CD28-stimulated T cell proliferation. Increasing concentrations of ibrutinib were evaluated: untreated (Figure 22A); 0.1 μM ibrutinib (Figure 22B); 0.5 μM ibrutinib (Figure 22C), 1 μM ibrutinib (Figure 22D), and 5 μM ibrutinib (Figure 22E).

[0131] [Figure 23] FIG. 23 is a graphic representation showing that ibrutinib does not affect CART19 cytotoxicity.

[0132] [Figure 24] FIG. 24 is a series of graphical representations showing that ibrutinib treatment does not promote TH1 / TH2 cytokine skewing in CART19 cells.

[0133] [Figure 25] Figures 25A and 25B are graphical representations showing that continuous administration of ibrutinib does not affect CART19 function in tumor cell elimination in vivo. Figure 25A shows Nalm / 6 cells detected in peripheral blood during each treatment regimen. Figure 25B shows Kaplan-Meier survival curves comparing survival of mice receiving CART19 with or without ibrutinib dosing.

[0134] [Figure 26]Figures 26A, 26B, 26C, 26D, 26E, and 26F are graphical representations showing the efficiency of CAR19 transduction in CLL patient cells at the indicated time points during ibrutinib treatment. Cells were untransduced (Figures 26A, 26B, and 26C) or transduced with CAR19 (Figures 26D, 26E, and 26F). Cells stained with GAM express CAR19 and are shown in the box in each profile.

[0135] [Figure 27-1] Figure 27 is a series of graphical representations depicting the proliferation rate of non-transduced cells compared to CAR19-transduced cells at indicated time points during ibrutinib treatment from a panel of patients. [Figure 27-2] Figure 27 is a series of graphical representations depicting the proliferation rate of non-transduced cells compared to CAR19-transduced cells at indicated time points during ibrutinib treatment from a panel of patients. [Figure 27-3] Figure 27 is a series of graphical representations depicting the proliferation rate of non-transduced cells compared to CAR19-transduced cells at indicated time points during ibrutinib treatment from a panel of patients.

[0136] [Figure 28] Figures 28A, 28B, and 28C are graphical representations showing that ibrutinib treatment induces lymphocytosis in CLL patients. Cells from patients were isolated at the indicated time points: baseline (Figure 28A); cycle 2, day 1 (Figure 28B); and cycle 12, day 1 (Figure 28C).

[0137] [Figure 29] Figures 29A, 29B, and 29C are graphical representations showing that ibrutinib treatment in three CLL patients reduces CD200 expression on tumor cells in CLL over time. Each profile contains an overlay of CD200 expression histograms from cells isolated at the specified time point: baseline (screen); cycle 2, day 1; and cycle 12, day 1.

[0138] [Figure 30] Figures 30A, 30B, and 30C are graphical representations showing that ibrutinib treatment in CLL patients reduces the frequency of PD1+ T cells over time. Cells from patients were isolated at the indicated time points: baseline (Figure 30A); cycle 2, day 1 (Figure 30B); and cycle 12, day 1 (Figure 30C).

[0139] [Figure 31] Figures 31A and 31B are graphic representations showing the sensitivity of MCL cell lines RL (Figure 31A) and JEKO-1 (Figure 31B) to ibrutinib treatment.

[0140] [Figure 32] FIG. 32 is a graphic representation showing the effect of ibrutinib treatment in an in vivo model of MCL.

[0141] [Figure 33] Figures 33A and B are images of immunohistochemical analysis of Hodgkin's lymphoma showing the presence of CD19-expressing cells in the tumor. Figure 33A is at 1x magnification and Figure 33B is at 20x magnification.

[0142] [Figure 34] FIG. 34 shows a schematic of the experimental procedure for a study evaluating the therapeutic efficacy of CART19 treatment in patients with Hodgkin's lymphoma.

[0143] [Figure 35-1]Figures 35A, 35B, 35C, and 35D show flow cytometry analysis of PD1 and CAR19 expression on T cells. Figures 35A and 35B are representative flow cytometry profiles showing the distribution of PD-1 and CAR19 expression on CD4+ T cells from subjects who were complete responders (CR) or non-responders (NR) to CART therapy. Figure 35C is a graph showing the percentage of PD1 cells in the CD4+ T cell population from multiple subject groups with different responses to CART therapy. Figure 35D is a graph showing the percentage of PD1 cells in the CD8+ T cell population from multiple subject groups with different responses to CART therapy. [Figure 35-2] Figures 35A, 35B, 35C, and 35D show flow cytometry analysis of PD1 and CAR19 expression on T cells. Figures 35A and 35B are representative flow cytometry profiles showing the distribution of PD-1 and CAR19 expression on CD4+ T cells from subjects who were complete responders (CR) or non-responders (NR) to CART therapy. Figure 35C is a graph showing the percentage of PD1 cells in the CD4+ T cell population from multiple subject groups with different responses to CART therapy. Figure 35D is a graph showing the percentage of PD1 cells in the CD8+ T cell population from multiple subject groups with different responses to CART therapy.

[0144] [Figure 36] Figures 36A and 36B show the distribution of PD1 expression in CD4- and CAR19-expressing cells (Figure 36A) or CD8- and CAR19-expressing cells (Figure 36B) from multiple subject groups with different responses to CART therapy.

[0145] [Figure 37] Figure 37 shows flow cytometry analysis of PD1, CAR 19, LAG3 and TIM3 expression in T cells from subjects who were complete responders (CR) or non-responders (NR) to CART treatment.

[0146] [Figure 38] Figures 38A and 38B show the distribution of PD1 and LAG3 expression (Figure 38A) or PD1 and TIM3 expression (Figure 38B) from multiple subject groups with different responses to CART treatment.

[0147] [Figure 39] Figure 39 shows plasma cell IgA immunophenotypes from myeloma patients receiving CART19, indicating response to CART19 treatment.

[0148] [Figure 40] Figures 40A and 40B are graphs showing the increase in titers against influenza vaccine strains compared to placebo. In Figure 40A, the increase above baseline in influenza geometric mean titers against each of the three influenza vaccine strains (H1N1 A / California / 07 / 2009, H3N2 A / Victoria / 210 / 2009, B / Brisbane / 60 / 2008) relative to the increase in the placebo cohort 4 weeks after vaccination is shown for each of the RAD001 dosing cohorts in the intention-to-treat population. The bold black line indicates a 1.2-fold increase in titer relative to placebo, which is required for two of the three influenza vaccine strains to meet the trial's primary endpoint. An asterisk "*" indicates that the GMT titer increase relative to placebo is greater than 1, with a posterior probability of at least 80%. FIG. 40B is a graph of the same data as in FIG. 40A for the subset of subjects with baseline influenza titers ≦1:40.

[0149] [Figure 41] Figure 41 is a scatter plot of RAD001 concentrations 4 weeks after vaccination versus the fold increase in geometric mean titers for each influenza vaccine strain. RAD001 concentrations (1 hour post-dose) were measured after subjects had been dosed for 4 weeks. All subjects who underwent pharmacokinetic measurements were included in the analysis set. The fold increase in geometric mean titers 4 weeks after vaccination relative to baseline is shown on the y-axis.

[0150] [Figure 42] Figure 42 is a graphical representation showing the increase in titers against heterologous influenza strains compared to placebo. The increase above baseline in influenza geometric mean titers against two heterologous influenza strains not included in the influenza vaccine (A / H1N1 strain A / New Jersey / 8 / 76 and A / H3N2 strain A / Victoria / 361 / 11) compared to the increase in the placebo cohort 4 weeks after vaccination is shown for each of the RAD001 dosing cohorts in the intention-to-treat population. * indicates that the increase in titer relative to placebo is greater than 1 and has a posterior probability of at least 80%.

[0151] [Figure 43] Figures 43A and 43B are graphical representations of IgG and IgM levels before and after influenza vaccination. Anti-A / H1N1 / California / 07 / 2009 influenza IgG and IgM levels were measured in serum obtained from subjects before and 4 weeks after influenza vaccination. No significant differences in anti-H1N1 influenza IgG and IgM levels from baseline to 4 weeks after vaccination were detected between the RAD001 and placebo cohorts (all p-values >0.05 by Kruskal-Wallis rank sum test).

[0152] [Figure 44]Figures 44A, 44B, and 44C are graphic representations of the decrease in the percent of PD-1 positive CD4 and CD8 and the increase in PD-1 negative CD4 T cells following treatment with RAD001. The percent of PD-1 positive CD4, CD8, and PD-1 negative CD4 T cells were determined by FACS analysis of PBMC samples at baseline, 6 weeks after study drug treatment (Week 6), and 6 weeks after discontinuation of study drug and 4 weeks after influenza vaccination (Week 12). Figure 44A shows that there was a significant decrease (-37.1 to -28.5%) in PD-1 positive CD4 T cells in cohorts receiving RAD001 at dose levels of 0.5 mg / day (n=25), 5 mg / week (n=29), and 20 mg / week (n=30) at week 12 compared to the placebo cohort (n=25), with p=0.002 (0.02), p=0.003 (q=0.03), and p=0.01 (q=0.05), respectively. Figure 44B shows that there was a significant decrease (-43.3 to -38.5%) in PD-1 positive CD8 T cells in cohorts receiving RAD001 (n=109) at dose levels of 0.5 mg / day (n=25), 5 mg / week (n=29), and 20 mg / week (n=30) at week 12 compared to the placebo cohort (n=25), with p=0.01 (0.05), p=0.007 (q=0.04), and p=0.01 (q=0.05), respectively. Figure 44C shows that there was a significant increase (3.0-4.9%) in PD-1 negative CD4 T cells in cohorts receiving RAD001 (n=109) at dose levels of 0.5 mg / day (n=25), 5 mg / week (n=29), and 20 mg / week (n=30) at week 12 compared to the placebo cohort (n=25), with p=0.0007 (0.02), p=0.03 (q=0.07), and p=0.03 (q=0.08), respectively.

[0153] [Figure 45]Figures 45A and 45B are graphical representations of the decrease in the percent of PD-1-positive CD4 and CD8 T cells and the increase in PD-1-negative CD4 T cells after RAD001 treatment, adjusting for differences in baseline PD-1 expression. The percent of PD-1-positive CD4, CD8, and PD-1-negative CD4 T cells was determined by FACS analysis of PBMC samples at baseline, 6 weeks after study drug treatment (Week 6), and 6 weeks after study drug discontinuation and 4 weeks after influenza vaccination (Week 12). Figure 45A shows a significant 30.2% decrease in PD-1+ CD4 T cells at Week 6 in the pooled RAD cohort (n=84) compared to the placebo cohort (n=25), p=0.03 (q=0.13). The decrease in PD-1-positive CD4 T cells at Week 12 in the pooled RAD cohort compared to the placebo cohort was 32.7%, p=0.05 (q=0.19). Figure 45B shows a significant 37.4% reduction in PD-1 positive CD8 T cells at week 6 in the pooled RAD cohort (n=84) compared to the placebo cohort (n=25), with p=0.008 (q=0.07). The reduction in PD-1 positive CD8 T cells at week 12 in the pooled RAD compared to the placebo cohort was 41.4%, with p=0.066 (q=0.21). Figures 45A and 45B represent the data in Figures 44A, 44B, and 44C, except that the different RAD001 dose groups in Figures 44A, 44B, and 44C have been pooled into a single RAD001 treatment group in Figures 45A and 45B.

[0154] [Figure 46] FIG. 46 shows increased activity and energy in elderly subjects in response to RAD001.

[0155] [Figure 47] Figures 47A and 47B depict the predicted effects of RAD001 on P70 S6K activity in cells. Figure 47A depicts P70 S6 kinase inhibition with weekly and daily high doses of RAD001, and Figure 47B depicts P70 S6 kinase inhibition with weekly low doses of RAD001.

[0156] [Figure 48] Figures 48A and 48B show IL-7 receptor (CD127) expression in cancer cell lines and CART cells. CD127 expression was measured by flow cytometry analysis in three cancer cell lines: RL (mantle cell lymphoma), JEKO (also known as Jeko-1, mantle cell lymphoma), and Nalm-6 (B-ALL) (Figure 48A). CD127 expression was measured by flow cytometry analysis in circulating CD3-positive (CART) cells injected into NSG mice (Figure 48B).

[0157] [Figure 49-1] Figures 49A, 49B, and 49C show the antitumor response after CART19 treatment and subsequent IL-7 treatment. NSG mice implanted with a luciferase-expressing mantle lymphoma cell line (RL-luc) on day 0 were treated with various doses of CART19 cells on day 6, and tumor burden was monitored. Mice were divided into four groups receiving no CART19 cells, 0.5 x 10 CART19 cells (CART19 0.5E6), 1 x 10 CART19 cells (CART19 1E6), or 2 x 10 CART19 cells (CART19 2E6). Tumor burden after CART treatment was monitored by detection of bioluminescence (mean BLI) (Figure 49A). Mice receiving 0.5x10 CART19 cells (CART19 0.5E6) or 1x10 CART19 cells (CART19 1E6) were randomized to receive or not receive recombinant human IL-7 (rhIL-7). Tumor burden, now represented by mean bioluminescence (BLI), was monitored in three mice from Figure 49A (#3827, #3829, and #3815, which received the initial CART19 dose indicated) treated with IL-7 starting on day 85 (Figure 49B). IL-7 was administered by IP injection three times weekly. Tumor burden, represented here by mean bioluminescence (BLI), was compared between mice that did not receive IL-7 (CTRL) and mice that received IL-7 treatment (IL-7) 85 days before (PRE) and 115 days after (POST) (Figure 49C). [Figure 49-2]Figures 49A, 49B, and 49C show the antitumor response after CART19 treatment and subsequent IL-7 treatment. NSG mice implanted with a luciferase-expressing mantle lymphoma cell line (RL-luc) on day 0 were treated with various doses of CART19 cells on day 6, and tumor burden was monitored. Mice were divided into four groups receiving no CART19 cells, 0.5 x 10 CART19 cells (CART19 0.5E6), 1 x 10 CART19 cells (CART19 1E6), or 2 x 10 CART19 cells (CART19 2E6). Tumor burden after CART treatment was monitored by detection of bioluminescence (mean BLI) (Figure 49A). Mice receiving 0.5x10 CART19 cells (CART19 0.5E6) or 1x10 CART19 cells (CART19 1E6) were randomized to receive or not receive recombinant human IL-7 (rhIL-7). Tumor burden, now represented by mean bioluminescence (BLI), was monitored in three mice from Figure 49A (#3827, #3829, and #3815, which received the initial CART19 dose indicated) treated with IL-7 starting on day 85 (Figure 49B). IL-7 was administered by IP injection three times weekly. Tumor burden, represented here by mean bioluminescence (BLI), was compared between mice that did not receive IL-7 (CTRL) and mice that received IL-7 treatment (IL-7) 85 days before (PRE) and 115 days after (POST) (Figure 49C).

[0158] [Figure 50] Figures 50A and 50B show T cell dynamics after IL-7 treatment. The level of human T cells detected in the blood was monitored in each of the mice that received IL-7 or the control mice (Figure 50A). The level of CART19 cells (CD3+ cells) detected in the blood was measured before (PRE) and 14 days after (day 14) IL-7 treatment (Figure 50B).

[0159] [Figure 51]Figure 51 shows the structures of two representative RCAR configurations. The antigen binding member comprises an antigen binding domain, a transmembrane domain, and a switch domain. The intracellular binding member exhibits a switch domain, a costimulatory signaling domain, and a primary signaling domain. The two configurations demonstrate that the first and second switch domains described herein can be in different orientations relative to the antigen binding member and the intracellular binding member. Other RCAR configurations are further described herein.

[0160] [Figure 52-1] Figure 52A is an image of a RL cell line. Figure 52B is a series of flow cytometry scattergrams showing CD19 and CD5 expression in RL primary and RL cell lines. Figure 52C is an image showing the t(11;14) translocation by fluorescence in situ hybridization (FISH). Figure 52D is a graph showing the IC50 (by percentage MTT conversion) of ibrutinib inhibition in various cell lines. Figure 52E is a series of images and graphs showing RL cell engraftment and resulting tumor burden in NOD-SCID-gamma chain knockout (NSG) mice. Figure 52F is a series of histological images showing the localization of MCL cells to various organs in mice. Figure 52G is a series of histological images of a mouse injected with MCL-RL cells. [Figure 52-2] Figure 52A is an image of a RL cell line. Figure 52B is a series of flow cytometry scattergrams showing CD19 and CD5 expression in RL primary and RL cell lines. Figure 52C is an image showing the t(11;14) translocation by fluorescence in situ hybridization (FISH). Figure 52D is a graph showing the IC50 (by percentage MTT conversion) of ibrutinib inhibition in various cell lines. Figure 52E is a series of images and graphs showing RL cell engraftment and resulting tumor burden in NOD-SCID-gamma chain knockout (NSG) mice. Figure 52F is a series of histological images showing the localization of MCL cells to various organs in mice. Figure 52G is a series of histological images of a mouse injected with MCL-RL cells. [Figure 52-3] Figure 52A is an image of a RL cell line. Figure 52B is a series of flow cytometry scattergrams showing CD19 and CD5 expression in RL primary and RL cell lines. Figure 52C is an image showing the t(11;14) translocation by fluorescence in situ hybridization (FISH). Figure 52D is a graph showing the IC50 (by percentage MTT conversion) of ibrutinib inhibition in various cell lines. Figure 52E is a series of images and graphs showing RL cell engraftment and resulting tumor burden in NOD-SCID-gamma chain knockout (NSG) mice. Figure 52F is a series of histological images showing the localization of MCL cells to various organs in mice. Figure 52G is a series of histological images of a mouse injected with MCL-RL cells. [Figure 52-4] Figure 52A is an image of a RL cell line. Figure 52B is a series of flow cytometry scattergrams showing CD19 and CD5 expression in RL primary and RL cell lines. Figure 52C is an image showing the t(11;14) translocation by fluorescence in situ hybridization (FISH). Figure 52D is a graph showing the IC50 (by percentage MTT conversion) of ibrutinib inhibition in various cell lines. Figure 52E is a series of images and graphs showing RL cell engraftment and resulting tumor burden in NOD-SCID-gamma chain knockout (NSG) mice. Figure 52F is a series of histological images showing the localization of MCL cells to various organs in mice. Figure 52G is a series of histological images of a mouse injected with MCL-RL cells. [Figure 52-5]Figure 52A is an image of a RL cell line. Figure 52B is a series of flow cytometry scattergrams showing CD19 and CD5 expression in RL primary and RL cell lines. Figure 52C is an image showing the t(11;14) translocation by fluorescence in situ hybridization (FISH). Figure 52D is a graph showing the IC50 (by percentage MTT conversion) of ibrutinib inhibition in various cell lines. Figure 52E is a series of images and graphs showing RL cell engraftment and resulting tumor burden in NOD-SCID-gamma chain knockout (NSG) mice. Figure 52F is a series of histological images showing the localization of MCL cells to various organs in mice. Figure 52G is a series of histological images of a mouse injected with MCL-RL cells. [Figure 52-6] Figure 52A is an image of a RL cell line. Figure 52B is a series of flow cytometry scattergrams showing CD19 and CD5 expression in RL primary and RL cell lines. Figure 52C is an image showing the t(11;14) translocation by fluorescence in situ hybridization (FISH). Figure 52D is a graph showing the IC50 (by percentage MTT conversion) of ibrutinib inhibition in various cell lines. Figure 52E is a series of images and graphs showing RL cell engraftment and resulting tumor burden in NOD-SCID-gamma chain knockout (NSG) mice. Figure 52F is a series of histological images showing the localization of MCL cells to various organs in mice. Figure 52G is a series of histological images of a mouse injected with MCL-RL cells.

[0161] [Figure 53-1]Figure 53A is a series of graphs showing the number of CD107a+ CART19 cells upon exposure to various MCL cell lines. Figure 53B is a series of graphs showing the amount of IL-2 and TNF-alpha produced by CART19 cells upon exposure to various MCL cell lines. Figure 53C is a graph showing the percent killing of various MCL cell lines by CART19 cells at various effector:target cell ratios. Figure 53D is a graph showing the amount of carboxyfluorescein succinimidyl ester (CFSE), an indicator of proliferation, in CART19 cells exposed to various MCL cell lines. Figure 53E is a series of graphs showing the percentage of T cells before and after expansion. Figure 53F is a series of graphs showing the percentage of untransduced or CAR-19-transduced T cells expressing or producing various biomolecules (e.g., cytokines). [Figure 53-2] Figure 53A is a series of graphs showing the number of CD107a+ CART19 cells upon exposure to various MCL cell lines. Figure 53B is a series of graphs showing the amount of IL-2 and TNF-alpha produced by CART19 cells upon exposure to various MCL cell lines. Figure 53C is a graph showing the percent killing of various MCL cell lines by CART19 cells at various effector:target cell ratios. Figure 53D is a graph showing the amount of carboxyfluorescein succinimidyl ester (CFSE), an indicator of proliferation, in CART19 cells exposed to various MCL cell lines. Figure 53E is a series of graphs showing the percentage of T cells before and after expansion. Figure 53F is a series of graphs showing the percentage of untransduced or CAR-19-transduced T cells expressing or producing various biomolecules (e.g., cytokines). [Figure 53-3]Figure 53A is a series of graphs showing the number of CD107a+ CART19 cells upon exposure to various MCL cell lines. Figure 53B is a series of graphs showing the amount of IL-2 and TNF-alpha produced by CART19 cells upon exposure to various MCL cell lines. Figure 53C is a graph showing the percent killing of various MCL cell lines by CART19 cells at various effector:target cell ratios. Figure 53D is a graph showing the amount of carboxyfluorescein succinimidyl ester (CFSE), an indicator of proliferation, in CART19 cells exposed to various MCL cell lines. Figure 53E is a series of graphs showing the percentage of T cells before and after expansion. Figure 53F is a series of graphs showing the percentage of untransduced or CAR-19-transduced T cells expressing or producing various biomolecules (e.g., cytokines). [Figure 53-4] Figure 53A is a series of graphs showing the number of CD107a+ CART19 cells upon exposure to various MCL cell lines. Figure 53B is a series of graphs showing the amount of IL-2 and TNF-alpha produced by CART19 cells upon exposure to various MCL cell lines. Figure 53C is a graph showing the percent killing of various MCL cell lines by CART19 cells at various effector:target cell ratios. Figure 53D is a graph showing the amount of carboxyfluorescein succinimidyl ester (CFSE), an indicator of proliferation, in CART19 cells exposed to various MCL cell lines. Figure 53E is a series of graphs showing the percentage of T cells before and after expansion. Figure 53F is a series of graphs showing the percentage of untransduced or CAR-19-transduced T cells expressing or producing various biomolecules (e.g., cytokines). [Figure 53-5]Figure 53A is a series of graphs showing the number of CD107a+ CART19 cells upon exposure to various MCL cell lines. Figure 53B is a series of graphs showing the amount of IL-2 and TNF-alpha produced by CART19 cells upon exposure to various MCL cell lines. Figure 53C is a graph showing the percent killing of various MCL cell lines by CART19 cells at various effector:target cell ratios. Figure 53D is a graph showing the amount of carboxyfluorescein succinimidyl ester (CFSE), an indicator of proliferation, in CART19 cells exposed to various MCL cell lines. Figure 53E is a series of graphs showing the percentage of T cells before and after expansion. Figure 53F is a series of graphs showing the percentage of untransduced or CAR-19-transduced T cells expressing or producing various biomolecules (e.g., cytokines).

[0162] [Figure 54A] Figure 54A is a series of images showing activation of interleukin-2-inducible T cell kinase (ITK) upon specific or nonspecific stimulation of CART19 cells. Figure 54B is a series of graphs showing CD107a surface expression (an indicator of degranulation), IL-2 production, and TNF-alpha production by CART19 cells in the presence of various concentrations of ibrutinib. Figure 54C is a series of histograms showing the amount of CFSE in CART19 cells exposed to various MCL cell lines in the presence of various concentrations of ibrutinib. Figure 54D is a series of graphs showing the expression or production of various cytokines and biomarkers as indicators of the Th1 or Th2 status of CART19 cells in combination with various concentrations of ibrutinib. Figure 54E is a series of graphs showing the percentage killing of various MCL cell lines by CART19 cells in combination with various concentrations of ibrutinib. Figure 54F is a bar graph showing the expression of various markers of intrinsic cytotoxic function of CART19 cells in combination with various concentrations of ibrutinib. [Figure 54B]Figure 54A is a series of images showing activation of interleukin-2-inducible T cell kinase (ITK) upon specific or nonspecific stimulation of CART19 cells. Figure 54B is a series of graphs showing CD107a surface expression (an indicator of degranulation), IL-2 production, and TNF-alpha production by CART19 cells in the presence of various concentrations of ibrutinib. Figure 54C is a series of histograms showing the amount of CFSE in CART19 cells exposed to various MCL cell lines in the presence of various concentrations of ibrutinib. Figure 54D is a series of graphs showing the expression or production of various cytokines and biomarkers as indicators of the Th1 or Th2 status of CART19 cells in combination with various concentrations of ibrutinib. Figure 54E is a series of graphs showing the percentage killing of various MCL cell lines by CART19 cells in combination with various concentrations of ibrutinib. Figure 54F is a bar graph showing the expression of various markers of intrinsic cytotoxic function of CART19 cells in combination with various concentrations of ibrutinib. [Figure 54C] Figure 54A is a series of images showing activation of interleukin-2-inducible T cell kinase (ITK) upon specific or nonspecific stimulation of CART19 cells. Figure 54B is a series of graphs showing CD107a surface expression (an indicator of degranulation), IL-2 production, and TNF-alpha production by CART19 cells in the presence of various concentrations of ibrutinib. Figure 54C is a series of histograms showing the amount of CFSE in CART19 cells exposed to various MCL cell lines in the presence of various concentrations of ibrutinib. Figure 54D is a series of graphs showing the expression or production of various cytokines and biomarkers as indicators of the Th1 or Th2 status of CART19 cells in combination with various concentrations of ibrutinib. Figure 54E is a series of graphs showing the percentage killing of various MCL cell lines by CART19 cells in combination with various concentrations of ibrutinib. Figure 54F is a bar graph showing the expression of various markers of intrinsic cytotoxic function of CART19 cells in combination with various concentrations of ibrutinib. [Figure 54D-1]Figure 54A is a series of images showing activation of interleukin-2-inducible T cell kinase (ITK) upon specific or nonspecific stimulation of CART19 cells. Figure 54B is a series of graphs showing CD107a surface expression (an indicator of degranulation), IL-2 production, and TNF-alpha production by CART19 cells in the presence of various concentrations of ibrutinib. Figure 54C is a series of histograms showing the amount of CFSE in CART19 cells exposed to various MCL cell lines in the presence of various concentrations of ibrutinib. Figure 54D is a series of graphs showing the expression or production of various cytokines and biomarkers as indicators of the Th1 or Th2 status of CART19 cells in combination with various concentrations of ibrutinib. Figure 54E is a series of graphs showing the percentage killing of various MCL cell lines by CART19 cells in combination with various concentrations of ibrutinib. Figure 54F is a bar graph showing the expression of various markers of intrinsic cytotoxic function of CART19 cells in combination with various concentrations of ibrutinib. [Figure 54D-2] Figure 54A is a series of images showing activation of interleukin-2-inducible T cell kinase (ITK) upon specific or nonspecific stimulation of CART19 cells. Figure 54B is a series of graphs showing CD107a surface expression (an indicator of degranulation), IL-2 production, and TNF-alpha production by CART19 cells in the presence of various concentrations of ibrutinib. Figure 54C is a series of histograms showing the amount of CFSE in CART19 cells exposed to various MCL cell lines in the presence of various concentrations of ibrutinib. Figure 54D is a series of graphs showing the expression or production of various cytokines and biomarkers as indicators of the Th1 or Th2 status of CART19 cells in combination with various concentrations of ibrutinib. Figure 54E is a series of graphs showing the percentage killing of various MCL cell lines by CART19 cells in combination with various concentrations of ibrutinib. Figure 54F is a bar graph showing the expression of various markers of intrinsic cytotoxic function of CART19 cells in combination with various concentrations of ibrutinib. [Figure 54E]Figure 54A is a series of images showing activation of interleukin-2-inducible T cell kinase (ITK) upon specific or nonspecific stimulation of CART19 cells. Figure 54B is a series of graphs showing CD107a surface expression (an indicator of degranulation), IL-2 production, and TNF-alpha production by CART19 cells in the presence of various concentrations of ibrutinib. Figure 54C is a series of histograms showing the amount of CFSE in CART19 cells exposed to various MCL cell lines in the presence of various concentrations of ibrutinib. Figure 54D is a series of graphs showing the expression or production of various cytokines and biomarkers as indicators of the Th1 or Th2 status of CART19 cells in combination with various concentrations of ibrutinib. Figure 54E is a series of graphs showing the percentage killing of various MCL cell lines by CART19 cells in combination with various concentrations of ibrutinib. Figure 54F is a bar graph showing the expression of various markers of intrinsic cytotoxic function of CART19 cells in combination with various concentrations of ibrutinib. [Figure 54F] Figure 54A is a series of images showing activation of interleukin-2-inducible T cell kinase (ITK) upon specific or nonspecific stimulation of CART19 cells. Figure 54B is a series of graphs showing CD107a surface expression (an indicator of degranulation), IL-2 production, and TNF-alpha production by CART19 cells in the presence of various concentrations of ibrutinib. Figure 54C is a series of histograms showing the amount of CFSE in CART19 cells exposed to various MCL cell lines in the presence of various concentrations of ibrutinib. Figure 54D is a series of graphs showing the expression or production of various cytokines and biomarkers as indicators of the Th1 or Th2 status of CART19 cells in combination with various concentrations of ibrutinib. Figure 54E is a series of graphs showing the percentage killing of various MCL cell lines by CART19 cells in combination with various concentrations of ibrutinib. Figure 54F is a bar graph showing the expression of various markers of intrinsic cytotoxic function of CART19 cells in combination with various concentrations of ibrutinib.

[0163] [Figure 55] Figure 55 is a schematic diagram of the in vivo mouse model experimental procedure to test the effect of CART19 and / or ibrutinib on MCL-RL-injected mice, where the readout is luminescence (an indicator of tumor cell number).

[0164] [Figure 56] Figure 56 is a schematic diagram of the in vivo mouse model experimental procedure to test the effect of CART19 and / or ibrutinib on MCL-RL-injected mice, where the readout is luminescence (an indicator of tumor cell number).

[0165] [Figure 57] Figure 57 is a series of graphs showing luminescence (a measure of tumor cell number) in mice after treatment with various concentrations of ibrutinib and overall survival after treatment.

[0166] [Figure 58] Figure 58 is a series of graphs showing luminescence (a measure of tumor cell number) in mice treated with ibrutinib or CART19 cells as well as overall survival after treatment.

[0167] [Figure 59] Figure 59 is a graph showing luminescence (a measure of tumor cell number) in mice after treatment with ibrutinib, untreated T cells, ibrutinib and untreated T cells, CART19 cells, and CART19 cells and ibrutinib.

[0168] [Figure 60] Figure 60 is a graph showing luminescence (a measure of tumor cell number) in mice after treatment with ibrutinib alone, CART19 cells alone, or the combination of ibrutinib and CART19 cells.

[0169] [Figure 61-1]Figure 61A is a series of graphs showing the levels of Th1 cytokines produced in mice treated with ibrutinib and / or CART19 cells. Figure 61B is a series of graphs showing the levels of Th2 cytokines produced in mice treated with ibrutinib and / or CART19 cells. Figure 61C is a graph showing the percentage of cells expressing the proliferation marker Ki67 in mice treated with CART19 cells or CART19 cells + ibrutinib. Figure 61D is a graph showing the percentage of cells expressing the anti-apoptotic marker BCL-2 in mice treated with CART19 cells or CART19 cells + ibrutinib. [Figure 61-2] Figure 61A is a series of graphs showing the levels of Th1 cytokines produced in mice treated with ibrutinib and / or CART19 cells. Figure 61B is a series of graphs showing the levels of Th2 cytokines produced in mice treated with ibrutinib and / or CART19 cells. Figure 61C is a graph showing the percentage of cells expressing the proliferation marker Ki67 in mice treated with CART19 cells or CART19 cells + ibrutinib. Figure 61D is a graph showing the percentage of cells expressing the anti-apoptotic marker BCL-2 in mice treated with CART19 cells or CART19 cells + ibrutinib. [Figure 61-3] Figure 61A is a series of graphs showing the levels of Th1 cytokines produced in mice treated with ibrutinib and / or CART19 cells. Figure 61B is a series of graphs showing the levels of Th2 cytokines produced in mice treated with ibrutinib and / or CART19 cells. Figure 61C is a graph showing the percentage of cells expressing the proliferation marker Ki67 in mice treated with CART19 cells or CART19 cells + ibrutinib. Figure 61D is a graph showing the percentage of cells expressing the anti-apoptotic marker BCL-2 in mice treated with CART19 cells or CART19 cells + ibrutinib. DETAILED DESCRIPTION OF THE INVENTION

[0170] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0171] The terms "a" and "an" mean one or more than one (i.e., at least one). By way of example, the term "an element" means one element or more than one element.

[0172] The term "about," when referring to a measurable value such as an amount, time, etc., is meant to encompass deviations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value, provided such variations are appropriate for the practice of the disclosed method.

[0173]

[0013] The term "chimeric antigen receptor" or alternatively "CAR" refers to a set of, generally in its simplest embodiment, two polypeptides which, when in an immune effector cell, provides the cell with specificity for a target cell, generally a cancer cell, and intracellular signal production. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule, as defined below. In some aspects, the set of polypeptides are contiguous to one another, e.g., on the same polypeptide chain (e.g., comprising a chimeric fusion protein). In some embodiments, the set of polypeptides are not contiguous to one another, e.g., on different polypeptide chains. In some embodiments, the set of polypeptides comprises a dimerization switch that can link the polypeptides to one another due to the presence of a dimerization molecule, e.g., linking the antigen binding domain to the intracellular signaling domain. In one aspect, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In some aspects, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In one aspect, the costimulatory molecule is chosen from a costimulatory molecule described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule.In one aspect, the CAR is a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

[0174] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within a cell to control cellular activity through a defined signaling pathway, either by producing second messengers or by functioning as an effector by responding to such messengers.

[0175] As used herein, the term "CD19" refers to the Cluster of Differentiation 19 protein, an antigenic determinant detectable on leukemia precursor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found under UniProt / Swiss-Prot Accession No. P15391, and the nucleotide sequence encoding human CD19 can be found under Accession No. NM_001178098. As used herein, "CD19" includes proteins containing mutations of full-length wild-type CD19, such as point mutations, fragments, insertions, deletions, and splice variants. CD19 is expressed in most B-cell lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Other cells that express CD19 are provided below in the definition of "diseases associated with CD19 expression." It is also an early marker of B cell precursors. See, e.g., Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one aspect, the antigen-binding portion of a CART recognizes and binds to an antigen within the extracellular domain of the CD19 protein. In one aspect, the CD19 protein is expressed on cancer cells.

[0176] As used herein, the term "CD20" refers to an antigenic determinant known to be detectable on B cells. Human CD20 is also referred to as transmembrane 4-domain, subfamily A, member 1 (MS4A1). Human and mouse amino acid and nucleic acid sequences are available in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD20 can be found in Accession Nos. NP_690605.1 and NP_068769.2, and nucleotide sequences encoding transcript variants 1 and 3 of human CD20 can be found in Accession Nos. NM_152866.2 and NM_021950.3, respectively. In one aspect, the antigen-binding portion of the CAR recognizes and binds to an antigen within the extracellular domain of the CD20 protein. In one aspect, the CD20 protein is expressed on a cancer cell.

[0177] As used herein, the term "CD22" refers to an antigenic determinant known to be detectable on leukemia precursor cells. Human and murine amino acid and nucleic acid sequences are available in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequences of human CD22 isoforms 1-5 are available under Accession Nos. NP 001762.2, NP 001172028.1, NP 001172029.1, NP 001172030.1, and NP 001265346.1, respectively, and the nucleotide sequences encoding human CD22 variants 1-5 are available under Accession Nos. NM 001771.3, NM 001185099.1, NM 001185100.1, NM 001185101.1, and NM 001278417.1, respectively. In one aspect, the antigen binding portion of the CAR recognizes and binds to an antigen within the extracellular domain of the CD22 protein. In one aspect, the CD22 protein is expressed on cancer cells.

[0178]

[0023] As used herein, the term "ROR1" refers to an antigenic determinant known to be detectable on leukemia precursor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequences of human ROR1 isoform 1 and 2 precursors are available at Accession Nos. NP_005003.2 and NP_001077061.1, respectively, and the mRNA sequences encoding them are available at Accession Nos. NM_005012.3 and NM_001083592.1, respectively. In one aspect, the antigen-binding portion of the CAR recognizes and binds to an antigen within the extracellular domain of the ROR1 protein. In one aspect, the ROR1 protein is expressed in cancer cells.

[0179] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. An antibody can be a tetramer of immunoglobulin molecules.

[0180] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), Fv fragments, scFv antibody fragments, disulfide-bridged Fv (sdFv), Fd fragments consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDR or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0181] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., by a synthetic linker, e.g., a short flexible polypeptide linker, and can be expressed as a single polypeptide chain, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, an scFv, as used herein, can have the VL and VH variable regions in either orientation, e.g., with respect to the N- and C-termini of the polypeptide, and can comprise VL-linker-VH or VH-linker-VL.

[0182] The portion of the CAR of the invention comprising an antibody or antibody fragment thereof can exist in a variety of forms in which the antigen-binding domain is expressed as part of a contiguous polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen-binding domain of the CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment comprising an scFv. The precise amino acid sequence boundaries of a CDR can be determined using any number of well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (the "Chothia" numbering scheme), or a combination thereof.

[0183] As used herein, the term "binding domain" or "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that comprises at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In certain embodiments, an antibody molecule is a multispecific antibody molecule, e.g., comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence in the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence in the plurality has binding specificity for a second epitope. In certain embodiments, a multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies have specificity for no more than two antigens. Bispecific antibody molecules are characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0184] The portion of the CAR of the invention comprising an antibody or antibody fragment thereof can exist in a variety of forms in which the antigen-binding domain is expressed as part of a contiguous polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen-binding domain of the CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment comprising an scFv.

[0185] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which usually determines the class to which the antibody belongs.

[0186] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0187] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed in a bacteriophage or yeast expression system. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein or amino acid sequence that specifies the antibody, where the DNA or amino acid sequence is available in the art and has been produced using well-known recombinant DNA or amino acid sequence technology.

[0188] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can act as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will appreciate that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response, therefore, encodes an "antigen" as the term is used herein. Furthermore, those skilled in the art will appreciate that an antigen need not necessarily be encoded solely by the full-length nucleotide sequence of a gene. The present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and it will be readily apparent that these nucleotide sequences can be arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It will be readily apparent that antigens can be synthetically produced, derived from biological samples, or macromolecules other than polypeptides. Such biological samples can include, but are not limited to, tissue samples, tumor samples, and fluids containing cells or other biological components.

[0189] The term "anti-cancer effect" refers to a biological effect that can be demonstrated by various means, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-cancer effect" can also be inherently demonstrated by the ability of peptides, polynucleotides, cells, and antibodies in preventing the development of cancer. The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0190] The term "autologous" refers to any material derived from the same individual into which it is subsequently reintroduced.

[0191] The term "allogeneic" refers to any material derived from a different animal of the same species as the one into which the material is being introduced. Two or more individuals are said to be allogeneic to one another if their genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently genetically different to interact antigenically.

[0192] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0193] The term "cancer" refers to a disease characterized by the uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "carcinoma" are used interchangeably herein, e.g., both terms include solid and liquid tumors, e.g., generalized or circulating. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors.

[0194] The term "disease associated with CD19 expression" includes diseases associated with CD19 expression or conditions associated with cells that express, or have expressed, CD19 at any time, including, for example, a proliferative disease such as a cancer or malignancy or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or preleukemia; or a non-cancer-related indication associated with cells that express CD19. For the avoidance of doubt, a disease associated with CD19 expression can include conditions associated with cells that once expressed CD19 but no longer express CD19 because, for example, CD19 expression has been downregulated, e.g., by treatment with a CD19-targeting molecule, e.g., a CD19 CAR. In one aspect, the cancer associated with CD19 expression is a hematological cancer. In one aspect, the hematological cancer is leukemia or lymphoma. In one aspect, cancers associated with expression of CD19 include, e.g., cancers and malignancies including, but not limited to, one or more acute leukemias, including, but not limited to, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), acute lymphoid leukemia (ALL); one or more chronic leukemias, including, but not limited to, chronic myeloid leukemia (CML), chronic lymphoid leukemia (CLL). Additional cancers or hematological conditions associated with expression of CD19 include, but are not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's hypergammaglobulinemia, and "preleukemia," a diverse collection of hematological conditions united by the insufficient production (or dysplasia) of myeloid blood cells. Additionally, diseases associated with CD19 expression include, but are not limited to, for example, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative disorders associated with CD19 expression.Non-cancer-related indications associated with CD19 expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, tumor antigen-expressing cells express, or have expressed at any time, mRNA encoding a tumor antigen. In some embodiments, tumor antigen-expressing cells produce tumor antigen proteins (e.g., wild-type or mutant), and the tumor antigen proteins may be present at normal or low levels. In some embodiments, tumor antigen-expressing cells produce detectable levels of tumor antigen proteins at one time, and subsequently produce substantially no detectable tumor antigen proteins.

[0195] The term "disease associated with B cell antigen expression" includes, but is not limited to, diseases associated with expression of one or more of CD19, CD20, CD22, or ROR1 or conditions associated with cells that express, or have expressed at any time, one or more of CD19, CD20, CD22, or ROR1, including, for example, a proliferative disease such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, myelodysplastic syndrome, or preleukemia; or a non-cancer-related indication associated with cells that express one or more of CD19, CD20, CD22, or ROR1. For the avoidance of doubt, a disease associated with B cell antigen expression may include conditions associated with cells that once expressed a B cell antigen but do not currently express the antigen because, for example, antigen expression has been downregulated, for example, by treatment with a molecule that targets the B cell antigen, e.g., a B cell-targeting CAR. The term "disease associated with B cell antigen expression" includes diseases associated with CD19 expression, as described herein.

[0196] The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in a CAR of the invention can be replaced with other amino acid residues from the same side chain family, and the modified CAR can be tested using the functional assays described herein.

[0197] The term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex or a CAR) to its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signaling event, such as, but not limited to, signaling by the TCR / CD3 complex or signaling through the appropriate NK receptor or signaling domain of the CAR. Stimulation can mediate altered expression of a molecule.

[0198] The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides cytoplasmic signaling sequences that regulate activation of the immune cell in a stimulatory manner for at least some aspect of the immune cell signaling pathway. In one aspect, the signal is a primary signal that is initiated, for example, by binding of the TCR / CD3 complex to an MHC molecule loaded with peptide and leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that act in a stimulatory manner contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Examples of ITAM-containing cytoplasmic signaling sequences that are particularly useful in the present invention include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In specific CARs of the invention, the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signaling sequence, e.g., the primary signaling sequence of CD3 zeta. In specific CARs of the invention, the primary signaling sequence of CD3 zeta is the sequence provided as SEQ ID NO: 17 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In specific CARs of the invention, the primary signaling sequence of CD3 zeta is the sequence provided as SEQ ID NO: 43 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0199] The term "antigen-presenting cell" or "APC" refers to a cell of the immune system, such as an accessory cell (e.g., a B cell, a dendritic cell, etc.), that displays foreign antigens complexed with major histocompatibility complexes (MHC) on its surface. T cells recognize these complexes using the T cell receptor (TCR). APCs process antigens and present them to T cells.

[0200] "Intracellular signaling domain," as that term is used herein, refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of a CAR-containing cell, e.g., a CART cell. For example, examples of immune effector function in a CART cell include cytolytic activity and helper activity, including cytokine secretion.

[0201] In some embodiments, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent stimulation. In some embodiments, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain can comprise the cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain can comprise the cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0202] The primary intracellular signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

[0203] The term "zeta" or alternatively "zeta chain," "CD3-zeta," or "TCR-zeta" is defined as the protein provided as GenBank Acc. No. BAG36664.1, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and a "zeta stimulatory domain" or alternatively a "CD3-zeta stimulatory domain" or a "TCR-zeta stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain, or a functional derivative thereof, that are sufficient for functional transmission of the initial signal required for T cell activation. In one aspect, the cytoplasmic domain of zeta comprises residues 52-164 of GenBank Acc. No. BAG36664.1, or the equivalent residues from a non-human species that is a functional ortholog, e.g., mouse, rodent, monkey, ape, etc. In one aspect, a "zeta stimulatory domain" or "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO: 17. In one aspect, the "zeta stimulatory domain" or "CD3 zeta stimulatory domain" is the sequence provided as SEQ ID NO:43.

[0204] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, These include ligands that specifically bind to LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0205] The costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and ligands that specifically bind to CD83.

[0206] The intracellular signaling domain can comprise the entire intracellular portion or the entire naturally occurring intracellular signaling domain of the molecule from which it is derived, or a functional fragment or derivative thereof.

[0207] The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and the "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In one aspect, the "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO: 16, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0208] "Immune effector cells," as the term is used herein, refer to cells that are involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0209] As used herein, the term "immune effector function or immune effector response" refers to a function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response refers to a property of a T cell or NK cell that promotes the killing of or inhibits the growth or proliferation of a target cell. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

[0210] The term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have a particular nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a particular amino acid sequence and the biological properties derived therefrom. Thus, a gene, cDNA, or RNA encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand and the non-coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0211] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The term nucleotide sequence encoding a protein or RNA can also include introns, to the extent that the nucleotide sequence that encodes the protein may, in some versions, contain introns.

[0212] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, preparation, substance or composition described herein effective to achieve a particular biological result.

[0213] The term "endogenous" refers to any substance that originates or is produced within an organism, cell, tissue, or system.

[0214] The term "exogenous" refers to any substance introduced into or produced outside of an organism, cell, tissue, or system.

[0215] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0216] The term "transfer vector" refers to a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including linear polynucleotides, polynucleotides bound to ionic or amphipathic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes self-replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0217] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0218] The term "lentivirus" refers to a genus in the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and they can deliver significant amounts of genetic information into the DNA of host cells, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0219] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including self-inactivating lentiviral vectors, particularly as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used in clinical settings include, for example, Oxford BioMedica's LENTIVECTOR® Gene Delivery Technology and Lentigen's LENTIMAX®. TM Vector systems include, but are not limited to, non-clinical lentiviral vectors, etc. Non-clinical lentiviral vectors are also available and known to those skilled in the art.

[0220] The term "homologous" or "identity" refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When the subunit position in both of the two molecules is occupied by the same monomeric subunit; for example, if a position in two DNA molecules is occupied by adenine, they are homologous at that position. The homology between two sequences is a linear function of the number of matching or homologous positions; for example, if half of the positions in two sequences (e.g., 5 positions in a polymer 10 subunits long) are homologous, the two sequences are 50% homologous, and if 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0221] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody or antibody fragment performance. Generally, a humanized antibody or antibody fragment thereof comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a substantial portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0222] The term "fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, which consists of an amino acid sequence identical to a human form of an antibody or immunoglobulin, the entire molecule of which is of human origin.

[0223] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials in its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or it can exist in a non-native environment, such as, for example, a host cell.

[0224] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" for adenosine, "C" for cytosine, "G" for guanosine, "T" for thymidine, and "U" for uridine.

[0225] The term "operably linked" or "transcriptional control" refers to a functional association between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, where necessary to link two protein-coding regions, in the same reading frame.

[0226] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, intratumoral injection or infusion techniques.

[0227] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly described. Specifically, degenerate codon substitution can be achieved by producing sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0228] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds comprising amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no upper limit to the number of amino acids that may be included in a protein or peptide sequence. A polypeptide refers to any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term includes both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, of which many types exist, commonly referred to in the art as proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, or combinations thereof.

[0229] The term "promoter" refers to a DNA sequence recognized by or introduced into the synthetic machinery of a cell necessary to initiate the specific transcription of a polynucleotide sequence.

[0230] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence, and in other instances, this sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that causes the gene product to be expressed in a tissue-specific manner.

[0231] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a cell to produce the gene product under most or all physiological conditions of the cell.

[0232] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a cell to produce the gene product substantially only when an inducer corresponding to the promoter is present in the cell.

[0233] The term "tissue-specific" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes a cell to produce a gene product substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0234] The term "flexible polypeptide linker" or "linker," as used in the context of scFvs, refers to a peptide linker composed of amino acids, such as glycine and / or serine residues, used alone or in combination to link the variable heavy and variable light chain regions. In some embodiments, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer greater than or equal to 1. For example, n=1, n=2, n=3, n=4, n=5, and n=6, n=7, n=8, n=9, and n=10 (SEQ ID NO: 105). In some embodiments, the flexible polypeptide linker includes, but is not limited to, (Gly4Ser)4 (SEQ ID NO: 106) or (Gly4Ser)3 (SEQ ID NO: 107). In other embodiments, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 108). Also included within the scope of the present invention are the linkers described in WO2012 / 138475, which is incorporated herein by reference.

[0235] The 5' cap used here (RNA cap, RNA 7-methylguanosine cap or RNA m 7 A 5' cap (also called a G-cap) is a modified guanine nucleotide added to the "front," or 5' end, of eukaryotic messenger RNA shortly after transcription initiation. The 5' cap consists of a terminal group attached to the first transcribed nucleotide. Its presence is important for recognition by ribosomes and protection from RNases. Capping occurs cotranscriptionally, coupled to transcription, with one affecting the other. Shortly after transcription initiation, the 5' end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions required for mRNA capping. Synthesis proceeds as a multistep biochemical reaction. The capping moiety can be modified to modulate mRNA functions such as stability or translation efficiency.

[0236] As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, that has been synthesized in vitro. Generally, in vitro transcribed RNA is produced from an in vitro transcription vector. The in vitro transcription vector contains a template that is used to produce the in vitro transcribed RNA.

[0237] As used herein, "poly(A)" refers to a series of adenosines attached to mRNA by polyadenylation. In a preferred embodiment of a transient expression construct, the poly(A) is 50-5000 (SEQ ID NO: 109), preferably greater than 64, more preferably greater than 100, and most preferably greater than 300 or 400. The poly(A) sequence can be chemically or enzymatically modified to modulate mRNA function, such as localization, stability, or translation efficiency.

[0238] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety or modified variants thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' ends. The 3' poly(A) tail is a long sequence (often several hundred) of adenine nucleotides added to pre-mRNA by the action of the enzyme polyadenylation polymerase. In higher eukaryotes, the poly(A) tail is added to transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and its associated proteins help protect the mRNA from exonucleolytic degradation. Polyadenylation is also important for transcription termination, mRNA export from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but can also occur later in the cytoplasm. After transcription is terminated, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0239] As used herein, "transient" refers to expression of a non-integrated transgene for a period of hours, days, or weeks, where the period of expression is shorter than the period of expression when the gene is integrated into the genome or contained in a stable plasmid replicon in the host cell.

[0240] The term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that are responsible for transmitting a signal from one part of a cell to another part of the cell. The term "cell surface receptor" includes molecules and complexes of molecules that can receive a signal and transmit the signal across the cell membrane.

[0241] The term "subject" is intended to include living organisms in which an immune response can be elicited (eg, mammals, humans).

[0242] The term "substantially purified" cells refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0243] As used herein, the term "therapy" refers to treatment. A therapeutic effect is achieved by reducing, suppressing, ameliorating, or eradicating a disease state.

[0244] As used herein, the term "prevention" refers to the preventative or protective treatment of a disease or disease state.

[0245] In the context of the present invention, a "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen that is common to a specific hyperproliferative disorder. In certain aspects, the hyperproliferative disorder antigens of the present invention are derived from cancers including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.

[0246] The terms "transfect" or "transformation" or "transduction" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0247] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a binding partner (e.g., a stimulatory tumor antigen) protein present in a sample, but which does not substantially recognize or bind to other molecules within the sample.

[0248] "Regulatable chimeric antigen receptor (RCAR)," as that term is used herein, refers to a set of, generally in its simplest embodiment, two polypeptides that, when present in an RCARX cell, provides the RCARX cell with specificity for a target cell, generally a cancer cell, and regulatable intracellular signal production or proliferation, which can optimize the immune effector properties of the RCARX cell. RCARX cells rely, at least in part, on an antigen-binding domain to provide specificity for target cells that contain an antigen bound by the antigen-binding domain. In some embodiments, the RCAR contains a dimerization switch that can link the intracellular signaling domain to the antigen-binding domain in the presence of a dimerization molecule.

[0249] A "membrane anchor" or "membrane tethering domain," as that term is used herein, refers to a polypeptide or moiety, e.g., a myristoyl group, sufficient to anchor an extracellular or intracellular domain to the plasma membrane.

[0250] The term "switch domain," as that term is used herein, e.g., when referring to an RCAR, refers to one that binds to another switch domain, generally a polypeptide-based one, in the presence of a dimerization molecule. Binding results in functional coupling of a first one linked to, e.g., fused to, a first switch domain with a second one linked to, e.g., fused to, a second switch domain. The first and second switch domains are collectively referred to as a dimerization switch. In some embodiments, the first and second switch domains are identical to each other, e.g., polypeptides with the same primary amino acid sequence, and are collectively referred to as a homodimerization switch. In some embodiments, the first and second switch domains are different from each other, e.g., polypeptides with different primary amino acid sequences, and are collectively referred to as a heterodimerization switch. In some embodiments, the switch is intracellular. In some embodiments, the switch is extracellular. In some embodiments, the switch domain is polypeptide-based, e.g., FKBP- or FRB-based, and the dimerization molecule is a small molecule, e.g., a rapalog. In some embodiments, the switch domain is polypeptide-based, e.g., an scFv that binds a myc peptide, and the dimerization molecule is a polypeptide, fragment thereof, or multimer of a polypeptide, e.g., a myc ligand or multimer of a myc ligand that binds one or more myc scFvs. In some embodiments, the switch domain is polypeptide-based, e.g., a myc receptor, and the dimerization molecule is an antibody or fragment thereof, e.g., a myc antibody.

[0251] As used herein, the term "dimerization molecule," e.g., when referring to RCAR, refers to a molecule that promotes association of a first switch domain and a second switch domain. In some embodiments, the dimerization molecule is not naturally present in a subject or is not present at concentrations that result in significant dimerization. In some embodiments, the dimerization molecule is a small molecule, e.g., rapamycin or a rapalog, e.g., RAD001.

[0252] The term "bioequivalent" refers to the amount of an agent, other than a reference compound (e.g., RAD001), required to produce an effect equivalent to that produced by a reference dose or amount of the reference compound (e.g., RAD001). In some embodiments, the effect is the level of mTOR inhibition, e.g., as measured by P70 S6 kinase inhibition, e.g., as assessed in an in vivo or in vitro assay, e.g., as measured by an assay described herein, e.g., a Boulay assay, or by measuring phosphorylated S6 levels by Western blot. In some embodiments, the effect is a change in the ratio of PD-1-positive / PD-1-negative T cells, as measured by cell sorting. In some embodiments, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of P70 S6 kinase inhibition as a reference dose or amount of a reference compound. In some embodiments, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of change in the ratio of PD-1-positive / PD-1-negative T cells as a reference dose or amount of a reference compound.

[0253] The term "low, immune-enhancing dose," when used in combination with an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR inhibitor, refers to a dose of the mTOR inhibitor that partially, but not completely, inhibits mTOR activity, e.g., as measured by inhibition of P70 S6 kinase activity. For example, methods for assessing mTOR activity by inhibition of P70 S6 kinase are described herein. The dose is insufficient to produce complete immune suppression, but sufficient to enhance the immune response. In some embodiments, the low, immune-enhancing dose of the mTOR inhibitor results in a decrease in the number of PD-1-positive T cells and / or an increase in the number of PD-1-negative T cells or an increase in the ratio of PD-1-negative T cells to PD-1-positive T cells. In some embodiments, the low, immune-enhancing dose of the mTOR inhibitor results in an increase in the number of naive T cells. In some embodiments, the low, immune-enhancing dose of the mTOR inhibitor results in one or more of the following: For example, increased expression of one or more of the following markers in memory T cells, e.g., memory T cell precursors: CD62L 高 , CD127 高 , CD27 + and BCL2; Decreased KLRG1 expression, e.g., in memory T cells, e.g., memory T cell precursors; and An increase in the number of memory T cell precursors, e.g., the number of cells with any one or combination of the following characteristics: CD62L 高 Increased CD127 高 Increased CD27 + increased, KLRG1 decreased and BCL2 increased; wherein the change occurs, for example, at least transiently, for example, as compared to an untreated subject.

[0254] As used herein, "refractory" refers to a disease, e.g., cancer, that does not respond to treatment. In some embodiments, a refractory cancer may be resistant to treatment before or at the onset of treatment. In other embodiments, a refractory cancer may become refractory during treatment.

[0255] As used herein, a "complete responder" refers to a subject with a disease, e.g., cancer, who exhibits a complete response, e.g., complete remission, to treatment. A complete response can be identified, for example, using the Cheson criteria described herein.

[0256] As used herein, a "partial responder" refers to a subject with a disease, e.g., cancer, who exhibits a partial response, e.g., a partial remission, to a treatment. A partial response can be identified, for example, using the Cheson criteria.

[0257] As used herein, a "non-responder" refers to a subject with a disease, e.g., cancer, that does not respond to treatment, e.g., the patient has stable disease or shows disease progression. Non-responders can be identified, for example, using the Cheson criteria described herein.

[0258] As used herein, the term "recurrence" refers to the reappearance of a disease (e.g., cancer) after an initial period of responsiveness (e.g., a complete or partial response). The initial period of responsiveness can include a decrease in cancer cell levels below a certain threshold, e.g., 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% or less. Reappearance can include an increase in cancer cell levels above a certain threshold, e.g., 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% or more. Recurrence can be identified, for example, using the Cheson criteria described herein.

[0259] Ranges: Throughout this specification, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be construed as specifically disclosing all the possible subranges as well as individual numerical values within that range. For example, description of a range such as 1 to 6 should be construed as specifically disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the scope.

[0260] overview Provided herein are compositions and methods for use in treating diseases such as cancer (e.g., hematological cancers or other B-cell malignancies) using immune effector cells (e.g., T cells or NK cells) expressing a chimeric antigen receptor (CAR) (e.g., a CAR that targets a B-cell marker such as CD19). The methods include, inter alia, administering immune effector cells (e.g., T cells or NK cells) expressing a B-cell-targeting CAR described herein in combination with other agents, such as kinase inhibitors, e.g., kinase inhibitors described herein.

[0261] The present invention provides, at least in part, experiments supporting the high efficacy of combining CAR therapy (e.g., B cell-targeted CAR therapy) with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib. Combining a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, with CAR therapy can increase the efficacy of the combined therapy over either kinase inhibitor monotherapy, CAR-expressing cell monotherapy, or both. These advantageous effects may, for example, allow for lower doses of the kinase inhibitor, CAR-expressing cells, or both while maintaining efficacy. The results herein are applicable to a wide range of cancers, e.g., hematological cancers and other B cell malignancies. For example, ibrutinib inhibits BTK, which is elevated in most lymphomas. CAR19-expressing immune effector cells (e.g., T cells or NK cells) target cancers with CD19 surface expression, which is expressed in most B cell malignancies. Any other B cell-targeting CAR (e.g., a CAR targeting one or more of CD20, CD22, or ROR1) can be used in place of or in combination with CAR19 in the combination therapy described herein. Thus, the combination of CAR therapy (e.g., one or more of CD19 CAR, CD20 CAR, CD22 CAR, or ROR1 CAR therapy) with a BTK inhibitor (e.g., ibrutinib) is suitable for treating a wide range of cancers involving B cell hyperproliferation, including lymphoma (e.g., Hodgkin's lymphoma), MCL, CLL, DLBCL, and multiple myeloma.

[0262] According to the present invention, ibrutinib can reduce tumor mass and migrate neoplastic B cells into the peripheral blood (see, for example, Example 8 herein). Without being bound by theory, some lymphomas, such as MCL, are characterized by clusters of cancerous cells in the proliferation centers of lymph nodes. CAR-expressing immune effector cells may have difficulty penetrating the densely packed clusters. Therefore, BTK inhibitors such as ibrutinib reduce tumor mass and migrate neoplastic B cells into the peripheral blood, making lymphoma cells more susceptible to attack by CAR-expressing cells.

[0263] Alternatively or in combination, BTK inhibitors such as ibrutinib also affect CAR-expressing cells. The present invention demonstrates that ibrutinib treatment increases circulating CART19 cell levels (see, e.g., the data presented in Example 8). Without being bound by theory, increased circulating CART19 cell levels may be the result of, for example, increased proliferation, altered T cell phenotype, or other factors. For example, ibrutinib can inhibit ITK, a kinase homologous to BTK. ITK is expressed in T cells, and its inhibition can alter T cell phenotype. Treatment with a kinase inhibitor such as ibrutinib can shift T cell phenotype from a Th2 to a Th1 phenotype, thereby increasing T cell proliferative capacity. Pretreatment or coadministration of a subject with a BTK inhibitor can increase T cell proliferative capacity in the subject, thereby increasing circulating CAR-expressing cell levels. Furthermore, subjects pretreated with a BTK inhibitor, e.g., ibrutinib, may have a T cell population with enhanced proliferative potential for apheresis for CAR production.

[0264] In some aspects, the present invention provides a number of chimeric antigen receptors (CARs) comprising an antibody or antibody fragment engineered to specifically bind to a B cell antigen (e.g., chosen from one or more of CD19, CD20, CD22, or ROR1 proteins). In some aspects, the present invention provides cells (e.g., T cells) engineered to express a CAR, wherein the CAR T cell ("CART") exhibits anti-cancer properties. In some aspects, the cell is transformed with a CAR, and the CAR is expressed on the cell surface. In some embodiments, the cell (e.g., T cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell may stably express the CAR. In other embodiments, the cell (e.g., T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding the CAR. In some such embodiments, the cell may transiently express the CAR.

[0265] In one aspect, the anti-CD19 protein binding portion of the CAR is an scFv antibody fragment. In one aspect, such antibody fragments are functional in that they retain equivalent binding affinity, e.g., they bind to the same antigen with equivalent affinity as the IgG antibody from which they were derived. In one aspect, such antibody fragments are functional in that they provide a biological response, including, but not limited to, activation of an immune response, inhibition of signal transduction originating from the target antigen, inhibition of kinase activity, etc., as will be understood by one of skill in the art. In one aspect, the anti-CD19 antigen binding domain of the CAR is an scFv antibody fragment that has been humanized compared to the murine sequence of the scFv from which it was derived. In one aspect, the parent murine scFv sequence is the CAR19 construct provided in PCT Publication WO2012 / 079000 (incorporated herein by reference) and provided herein as SEQ ID NO: 59. In one embodiment, the anti-CD19 binding domain is the scFv described in WO2012 / 079000 and provided in SEQ ID NO: 59.

[0266]

[00130] In one aspect, an antibody of the invention is incorporated into a chimeric antigen receptor (CAR). In one aspect, the CAR comprises the polypeptide sequence provided as SEQ ID NO: 12 in PCT Publication WO2012 / 079000, and provided herein as SEQ ID NO: 58, wherein the scFv domain is replaced by one or more sequences selected from SEQ ID NOs: 1-12. In one aspect, the scFv domain of SEQ ID NOs: 1-12 is a humanized variant of the scFv domain of SEQ ID NO: 59, an scFv fragment of murine origin that specifically binds to human CD19. Humanization of this murine scFv may be desirable in clinical settings, where murine-specific residues may elicit a human anti-mouse antigen (HAMA) response in patients undergoing CART19 treatment, e.g., treatment with T cells transduced with a CAR19 construct.

[0267] In one aspect, the anti-CD19 binding domain portion of the CAR of the invention, e.g., the humanized scFv portion, is encoded by a transgene whose sequence has been codon-optimized for expression in mammalian cells. In one aspect, the entire CAR construct of the invention is encoded by a transgene whose entire sequence has been codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that encode the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. Various codon optimization methods are known in the art and include, for example, the methods disclosed in at least U.S. Patent Nos. 5,786,464 and 6,114,148.

[0268] In one aspect, the humanized CAR19 comprises an scFv portion provided in SEQ ID NO: 1. In one aspect, the humanized CAR19 comprises an scFv portion provided in SEQ ID NO: 2. In one aspect, the humanized CAR19 comprises an scFv portion provided in SEQ ID NO: 3. In one aspect, the humanized CAR19 comprises an scFv portion provided in SEQ ID NO: 4. In one aspect, the humanized CAR19 comprises an scFv portion provided in SEQ ID NO: 5. In one aspect, the humanized CAR19 comprises an scFv portion provided in SEQ ID NO: 6. In one aspect, the humanized CAR19 comprises an scFv portion provided in SEQ ID NO: 7. In one aspect, the humanized CAR19 comprises an scFv portion provided in SEQ ID NO: 8. In one aspect, the humanized CAR19 comprises an scFv portion provided in SEQ ID NO: 9. In one aspect, the humanized CAR19 comprises an scFv portion provided in SEQ ID NO: 10. In one aspect, the humanized CAR19 comprises an scFv portion provided in SEQ ID NO: 11. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO: 12.

[0269]

[0013] In one aspect, a CAR of the present invention combines the antigen binding domain of a specific antibody with an intracellular signaling molecule. For example, in one aspect, the intracellular signaling molecule includes, but is not limited to, the CD3 zeta chain, 4-1BB, and CD28 signaling modules, and combinations thereof. In one aspect, a CD19 CAR comprises a CAR selected from the sequence provided in one or more of SEQ ID NOs: 31-42. In one aspect, a CD19 CAR comprises the sequence provided in SEQ ID NO: 31. In one aspect, a CD19 CAR comprises the sequence provided in SEQ ID NO: 32. In one aspect, a CD19 CAR comprises the sequence provided in SEQ ID NO: 33. In one aspect, a CD19 CAR comprises the sequence provided in SEQ ID NO: 34. In one aspect, a CD19 CAR comprises the sequence provided in SEQ ID NO: 35. In one aspect, a CD19 CAR comprises the sequence provided in SEQ ID NO: 36. In one aspect, a CD19 CAR comprises the sequence provided in SEQ ID NO: 37. In one aspect, a CD19 CAR comprises the sequence provided in SEQ ID NO: 38. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO: 39. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO: 40. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO: 41. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO: 42.

[0270] Additionally, the present invention provides CD19 CAR compositions and their use in medicaments or methods for treating, among other diseases, cancer or any malignancy or autoimmune disease involving cells or tissues expressing CD19.

[0271] In one aspect, the CARs of the invention can be used to eradicate CD19-expressing normal cells, thereby making them applicable for use as a cell conditioning therapy prior to cell transplantation. In one aspect, the CD19-expressing normal cells are CD19-expressing normal stem cells, and the cell transplantation is stem cell transplantation.

[0272] In one aspect, the present invention provides cells (e.g., T cells) engineered to express a chimeric antigen receptor (CAR), wherein the CAR-expressing cells, e.g., CAR T cells ("CART"), exhibit anti-cancer properties. A preferred antigen is CD19. In one aspect, the antigen binding domain of the CAR comprises a partially humanized anti-CD19 antibody fragment. In one aspect, the antigen binding domain of the CAR comprises a partially humanized anti-CD19 antibody fragment comprising an scFv. Accordingly, the present invention provides CD19-CARs comprising a humanized anti-CD19 binding domain and engineered into immune effector cells, e.g., T cells or NK cells, and methods of use thereof for adoptive therapy.

[0273] In one aspect, the CD19-CAR comprises at least one intracellular domain selected from the group consisting of a CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3 zeta signal domain, and any combination thereof. In one aspect, the CD19-CAR comprises at least one intracellular signaling domain from one or more costimulatory molecules other than CD137 (4-1BB) or CD28.

[0274] Chimeric antigen receptor (CAR) The present invention encompasses recombinant DNA constructs comprising a sequence encoding a CAR, wherein the sequence comprises an antibody or antibody fragment that specifically binds to a CAR B cell antigen (e.g., CD19, e.g., human CD19), wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. The intracellular signaling domain may comprise a costimulatory signaling domain and / or a primary signaling domain, e.g., a zeta chain. The costimulatory signaling domain refers to a portion of a CAR that comprises at least a portion of the intracellular domain of a costimulatory molecule. In some embodiments, the antigen-binding domain is a murine antibody or antibody fragment described herein. In some embodiments, the antigen-binding domain is a humanized antibody or antibody fragment.

[0275] In a specific aspect, a CAR construct of the invention comprises an scFv domain selected from the group consisting of SEQ ID NOs: 1-12 or an scFV domain of SEQ ID NO: 59, wherein the scFv can be preceded by an optional leader sequence such as provided in SEQ ID NO: 13 and followed by an optional hinge sequence such as provided in SEQ ID NO: 14 or SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49, a transmembrane region such as provided in SEQ ID NO: 15, an intracellular signaling domain comprising SEQ ID NO: 16 or SEQ ID NO: 51, and a CD3 zeta sequence comprising SEQ ID NO: 17 or SEQ ID NO: 43, wherein the domains are contiguous and in the same reading frame to form a single fusion protein. Also encompassed by the invention are nucleotide sequences encoding each polypeptide of an scFv fragment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 59. Also encompassed by the present invention are nucleotide sequences encoding each polypeptide of an scFv fragment selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:59, and each domain of SEQ ID NOs:13-17, and the encoded CD19CAR fusion proteins of the present invention. In one aspect, an exemplary CD19CAR construct comprises an optional leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain. In one aspect, an exemplary CD19CAR construct comprises an optional leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, an intracellular costimulatory domain, and an intracellular stimulatory domain. Specific CD19 CAR constructs comprising a humanized scFv domain of the present invention are provided as SEQ ID NOs:31-42, or the murine scFv domain provided as SEQ ID NO:59.

[0276] The full-length CAR sequences are also provided herein as SEQ ID NOs: 31-42 and 58, as shown in Tables 7 and 3.

[0277] An exemplary leader sequence is provided as SEQ ID NO: 13. An exemplary hinge / spacer sequence is provided as SEQ ID NO: 14 or SEQ ID NO: 45 or SEQ ID NO: 47 or SEQ ID NO: 49. An exemplary transmembrane domain sequence is provided as SEQ ID NO: 15. An exemplary sequence of the intracellular signaling domain of 4-1BB protein is provided as SEQ ID NO: 16. An exemplary sequence of the intracellular signaling domain of CD27 is provided as SEQ ID NO: 51. An exemplary CD3 zeta domain sequence is provided as SEQ ID NO: 17 or SEQ ID NO: 43.

[0278] In one aspect, the invention encompasses a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an anti-CD19 binding domain, e.g., as described herein, that is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. In one aspect, the anti-CD19 binding domain is selected from one or more of SEQ ID NOs: 1-12 and 58. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64-813 of a sequence provided in one or more of SEQ ID NOs: 61-72 and 59. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 61. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 62. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 63. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64-813 of SEQ ID NO: 64. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO: 65. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO: 66. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO: 67. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO: 68. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO: 69. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO: 70. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO: 71. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO: 72.

[0279] In one aspect, the present invention encompasses a recombinant nucleic acid construct comprising a transgene encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding one or more anti-CD19 binding domains selected from SEQ ID NOs: 61-72, wherein the sequence is contiguous with and in the same reading frame as the nucleic acid sequence encoding the intracellular signaling domain. Exemplary intracellular signaling domains that can be used in a CAR include, but are not limited to, one or more intracellular signaling domains, such as, for example, CD3 zeta, CD28, 4-1BB, and the like. In some examples, a CAR may comprise any combination of CD3 zeta, CD28, 4-1BB, and the like. In one aspect, the nucleic acid sequence of a CAR construct of the invention is one or more selected from SEQ ID NOs: 85-96. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO: 85. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO: 86. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO: 87. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO: 88. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO: 89. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 90. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 91. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 92. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 93. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 94. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 95. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 96. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 97. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 98. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 99.

[0280] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as screening libraries from cells which express the gene, deriving the gene from a vector known to contain it, or isolating it directly from cells and tissues which contain it, using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically, rather than cloned.

[0281] The present invention includes retroviral and lentiviral vector constructs expressing CARs that can be directly transduced into cells.

[0282] The present invention also includes RNA constructs that can be directly transfected into cells. Methods for producing mRNA for use in transfection include in vitro transcription (IVT) of a template using specially designed primers, followed by poly(A) addition, to produce a construct typically 50-2000 bases in length, containing 3' and 5' untranslated sequences ("UTR"), a 5' cap and / or internal ribosome entry site (IRES), the gene to be expressed, and a poly(A) tail (SEQ ID NO: 118). The RNA thus produced efficiently transfects cells of different species. In some embodiments, the template contains the sequence of the CAR. In some embodiments, the RNA CAR vector is transduced into T cells by electroporation.

[0283] antigen-binding domain In one aspect, the CAR of the present invention comprises a target-specific binding element, otherwise referred to as an antigen-binding domain. The selection of the portion depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain can be selected to recognize a ligand that acts as a cell surface marker on the target cell associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain in the CAR of the present invention include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0284] In one aspect, a CAR-mediated T cell response can be directed to an antigen of interest by engineering an antigen binding domain into the CAR that specifically binds to the desired antigen.

[0285] In one aspect, the portion of the CAR comprising the antigen binding domain comprises an antigen binding domain that targets CD19. In one aspect, the antigen binding domain targets human CD19. In one aspect, the antigen binding domain of the CAR has the same or similar binding specificity as the FMC63 scFv fragment described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one embodiment, the antigen binding domain of the CAR comprises the scFv fragment described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997).

[0286] The antigen binding domain may be any domain that binds to an antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, murine antibodies, human antibodies, humanized antibodies and functional fragments thereof, including but not limited to single domain antibodies such as heavy chain variable domains (VH), light chain variable domains (VL) and variable domains of camelid-derived nanobodies (VHH), and alternative scaffolds known to function as antigen binding domains, such as recombinant fibronectin domains.

[0287] In one embodiment, the CAR molecule comprises an anti-CD19 binding domain comprising one or more (e.g., all three) light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of an anti-CD19 binding domain described herein, and one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of an anti-CD19 binding domain described herein, e.g., an anti-CD19 binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of an anti-CD19 binding domain described herein, e.g., the anti-CD19 binding domain has two variable heavy chain regions, each comprising an HC CDR1, HC CDR2, and HC CDR3 described herein. In one embodiment, the anti-CD19 binding domain comprises a murine light chain variable region described herein (e.g., in Table 7) and / or a murine heavy chain variable region described herein (e.g., in Table 7). In one embodiment, the anti-CD19 binding domain is an scFv comprising a murine light chain and a murine heavy chain of the amino acid sequences in Table 7. In one embodiment, the anti-CD19 binding domain (e.g., scFv) comprises a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided in Table 7, or a sequence with 95-99% identity to an amino acid sequence of Table 7; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided in Table 7, or a sequence with 95-99% identity to an amino acid sequence of Table 7. In one embodiment, the anti-CD19 binding domain comprises a sequence of SEQ ID NO: 59, or a sequence with 95-99% identity thereto.In one embodiment, the anti-CD19 binding domain is an scFv, in which a light chain variable region comprising an amino acid sequence described herein, e.g., in Table 7, is linked to a heavy chain variable region comprising an amino acid sequence described herein, e.g., in Table 7, via a linker, e.g., a linker described herein. In one embodiment, the anti-CD19 binding domain comprises a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53). The light chain variable region and heavy chain variable region of the scFv can be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0288] In some instances, it is advantageous for the antigen-binding domain to be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, it may be advantageous for the antigen-binding domain of the CAR to have human or humanized residues for the antigen-binding domain of an antibody or antibody fragment.

[0289] Thus, in one aspect, the antigen binding domain comprises a humanized antibody or antibody fragment. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of a murine or humanized anti-CD19 binding domain described herein and / or one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a murine or humanized anti-CD19 binding domain described herein, e.g., an anti-CD19 binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a murine or humanized anti-CD19 binding domain described herein, e.g., the humanized anti-CD19 binding domain has two variable heavy chain regions, each comprising an HC CDR1, an HC CDR2, and an HC CDR3 described herein. In one embodiment, the humanized anti-CD19 binding domain comprises a humanized light chain variable region described herein (e.g., in Table 3) and / or a humanized heavy chain variable region described herein (e.g., in Table 3). In one embodiment, the humanized anti-CD19 binding domain comprises a humanized heavy chain variable region described herein (e.g., in Table 3), e.g., at least two humanized heavy chain variable regions described herein (e.g., in Table 3). In certain embodiments, the anti-CD19 binding domain is an scFv comprising a light chain and a heavy chain of the amino acid sequences in Table 3.In one embodiment, the anti-CD19 binding domain (e.g., scFv) comprises a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided in Table 3, or a sequence with 95 to 99% identity to an amino acid sequence of Table 3; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided in Table 3, or a sequence with 95 to 99% identity to an amino acid sequence of Table 3. In one embodiment, the humanized anti-CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, or a sequence with 95 to 99% identity thereto. In one embodiment, the nucleic acid sequence encoding the humanized anti-CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, or a sequence having 95 to 99% identity thereto. In one embodiment, the humanized anti-CD19 binding domain is an scFv, in which a light chain variable region comprising an amino acid sequence described herein, e.g., in Table 3, is linked to a heavy chain variable region comprising an amino acid sequence described herein, e.g., in Table 3, via a linker, e.g., a linker described herein. In one embodiment, the humanized anti-CD19 binding domain comprises a (Gly4-Ser)n linker (where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4) (SEQ ID NO: 53). The light chain variable region and heavy chain variable region of the scFv may be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0290] In one aspect, the antigen binding domain portion comprises one or more sequences selected from SEQ ID NOs: 1-12. In one aspect, the humanized CAR is selected from one or more sequences selected from SEQ ID NOs: 31-42. In one aspect, the non-human antibody is humanized, where particular sequences or regions of the antibody have been modified to increase similarity to antibodies or fragments thereof that are naturally produced in humans.

[0291] Humanized antibodies can be produced by techniques such as CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089, which are incorporated herein by reference in their entireties), veneering, or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596, which are incorporated herein by reference in their entireties; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973), chain shuffling (see, for example, U.S. Patent No. 5,565,332, the entire contents of which are incorporated herein by reference), and, for example, U.S. Patent Application Publication No. US2005 / 0042664, U.S. Patent Application Publication No. US2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994) and Pedersen et al., J. Mol. Biol.CDRs can be generated using a variety of techniques known in the art, including, but not limited to, those disclosed in J. Am. Chem. Soc., 235(3):959-73 (1994). Often, framework residues in the framework regions are replaced with corresponding residues from the CDR donor antibody to alter, e.g., improve, antigen binding. These framework substitutions are made by methods well known in the art, for example, by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at specific positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, both of which are incorporated herein by reference in their entireties).

[0292] A humanized antibody or antibody fragment retains one or more amino acid residues from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. As provided herein, a humanized antibody or antibody fragment comprises one or more CDRs from a non-human immunoglobulin molecule and framework regions, where the amino acid residues making up the framework are derived entirely or predominantly from human germline. Techniques for humanizing antibodies or antibody fragments are well known in the art and can be performed essentially according to Winter et al. (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239,400; PCT Publication WO 91 / 09967; and U.S. Patent Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference in their entireties). In such humanized antibodies and antibody fragments, substantially less than an entire human variable domain is substituted with the corresponding sequence from a non-human species. Humanized antibodies are often human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted with residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Patent No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

[0293] The selection of human variable domains, both light and heavy, for producing humanized antibodies is intended to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)—the contents of which are incorporated herein by reference in their entirety). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (see, e.g., Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference in their entireties). In some embodiments, the framework regions of the heavy chain variable region, e.g., all four framework regions, are derived from the VH4_4-59 germline sequence. In some embodiments, the framework regions can contain one, two, three, four, or five modifications, e.g., substitutions, from, e.g., amino acids of the corresponding murine sequence (e.g., SEQ ID NO: 59). In some embodiments, the framework regions, e.g., all four framework regions of the light chain variable region, are derived from the VK3_1.25 germline sequence. In some embodiments, the framework regions can include, for example, 1, 2, 3, 4, or 5 modifications, eg, substitutions, from the amino acids of the corresponding murine sequence (eg, SEQ ID NO: 59).

[0294] In one aspect, the portion of the CAR composition of the present invention, including an antibody fragment, is humanized while retaining high affinity for the target antigen and other favorable biological properties. According to one aspect of the present invention, humanized antibodies and antibody fragments are produced by a process of analysis of the parent sequences and various conceptual humanized products using three-dimensional models of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs are available that model and display probable conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the potential role of residues in the function of the candidate immunoglobulin sequence, for example, analysis of residues that affect the ability of the candidate immunoglobulin to bind to the target antigen. In this way, FR residues from the recipient and import sequences can be selected and combined to achieve desired antibody or antibody fragment properties, such as increased affinity for the target antigen. Generally, CDR residues are directly and most substantially involved in influencing antigen binding.

[0295] A humanized antibody or antibody fragment may maintain antigenic specificity similar to that of the original antibody, e.g., in the present invention, the ability to bind to human CD19. In some embodiments, a humanized antibody or antibody fragment may have improved affinity and / or specificity of binding to human CD19.

[0296] In one aspect, the anti-CD19 binding domain is characterized by a particular functional attribute or property of an antibody or antibody fragment. For example, in one aspect, the portion of the CAR composition of the invention comprising the antigen binding domain specifically binds human CD19. In one aspect, the antigen binding domain has the same or a similar binding specificity for human CD19 as the FMC63 scFv described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one aspect, the invention relates to an antigen binding domain comprising an antibody or antibody fragment, wherein the antibody binding domain specifically binds to a CD19 protein or a fragment thereof, wherein the antibody or antibody fragment comprises a variable light chain and / or a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 1-12 or SEQ ID NO: 59. In one aspect, the antigen binding domain comprises the amino acid sequence of an scFv selected from SEQ ID NO: 1-12 or SEQ ID NO: 59. In one aspect, the scFv is flanked by a leader sequence and is in the same reading frame. In one aspect, the leader sequence is the polypeptide sequence provided as SEQ ID NO:13.

[0297] In one aspect, the anti-CD19 binding domain is a fragment, e.g., a single-chain variable fragment (scFv). In one aspect, the anti-CD19 binding domain is an Fv, Fab, (Fab')2, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In one aspect, the antibodies and fragments thereof of the present invention bind to the CD19 protein with wild-type or enhanced affinity.

[0298] In some instances, scFvs can be produced according to methods known in the art (see, e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking the VH and VL domains together using a flexible polypeptide linker of optimized length and / or amino acid composition (e.g., a Ser-Gly linker). Linker length can significantly affect how the variable regions of an scFv fold and interact. In fact, if a short polypeptide linker (e.g., 5-10 amino acids) is used, intrachain folding is prevented. Interchain folding is also required to bring two variable regions together to form a functional epitope-binding site. For examples of linker orientations and sizes, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Publication Nos. WO2006 / 020258 and WO2007 / 024715, which are incorporated herein by reference.

[0299] An scFv can include a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between the VL and VH regions. The linker sequence can include any naturally occurring amino acid. In some embodiments, the linker sequence includes the amino acids glycine and serine. In other embodiments, the linker sequence includes a set of glycine and serine repeats, such as (Gly4Ser)n (where n is a positive integer greater than or equal to 1) (SEQ ID NO: 18). In some embodiments, the linker can be (Gly4Ser)4 (SEQ ID NO: 106) or (Gly4Ser)3 (SEQ ID NO: 107). Varying the linker length can result in superior efficacy in activity tests, or can retain or enhance activity.

[0300] In some embodiments, the amino acid sequence of the antigen binding domain (or other portion, or the entire CAR) can be modified, e.g., the amino acid sequences described herein can be modified, e.g., by conservative substitutions. Families of amino acid residues having similar side chains have been defined in the art, and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0301] Percent identity in two or more nucleic acid or polypeptide sequences refers to two or more sequences that are the same. Two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identical, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection: Optionally, the identity exists over a region of at least about 50 nucleotides (or 10 amino acids) or more in length, preferably over a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0302] For sequence comparison, one sequence generally serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence alignment is designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computer-controlled implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology).

[0303] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0304] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4:11-17, as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can also be determined using the Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453 algorithm, as incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossom 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0305] In some aspects, the present invention contemplates modifications of the amino acid sequence of a starting antibody or fragment (e.g., scFv) to produce functionally equivalent molecules. For example, the VH or VL of an anti-CD19 binding domain, e.g., an scFv, comprised in a CAR can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the anti-CD19 binding domain, e.g., an scFv, of the starting VH or VL framework region. The present invention contemplates modifications of the entire CAR construct, e.g., modifications of one or more amino acid sequences of various domains of the CAR construct, to produce functionally equivalent molecules. The CAR construct can be modified to maintain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the starting CAR construct.

[0306] Bispecific CAR In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody molecule comprises heavy chain variable domain and light chain variable domain sequences that have binding specificity for a first epitope and heavy chain variable domain and light chain variable domain sequences that have binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In some embodiments, a bispecific antibody molecule comprises an scFv, or fragment thereof, having binding specificity for a first epitope and an scFv, or fragment thereof, having binding specificity for a second epitope.

[0307] Transmembrane domain With regard to the transmembrane domain, in various embodiments, a CAR can be designed to include a transmembrane domain that is linked to the extracellular domain of the CAR. The transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids that are linked to the extracellular region of the protein from which the transmembrane domain was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more additional amino acids that are linked to the intracellular region of the protein from which the transmembrane protein was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is linked to other domains of the CAR; for example, in one embodiment, the transmembrane domain can be derived from the same protein from which the signaling domain, costimulatory domain, or hinge domain was derived. In other aspects, the transmembrane domain is not derived from the same protein from which any other domain of the CAR is derived. In certain instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain can homodimerize with other CARs on the cell surface of a CAR-expressing cell. In a different aspect, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interactions with the binding domains of natural binding partners present on the same CAR-expressing cell.

[0308] The transmembrane domain may be naturally derived or derived from a recombinant source. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain can transmit signals to the intracellular domain when the CAR binds to the target. Transmembrane domains particularly useful in the present invention may include at least the transmembrane region of, for example, the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the transmembrane domain is selected from the group consisting of, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITG It may contain at least the transmembrane domains of AM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, and NKG2C.

[0309] In some examples, the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the antigen-binding domain of the CAR, via a hinge, e.g., a hinge from a human protein. For example, in some embodiments, the hinge can be a human Ig (immunoglobulin) hinge, e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge, or a CD8a hinge. In some embodiments, the hinge or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO: 14. In one aspect, the transmembrane domain comprises (e.g., consists of) the amino acid sequence of SEQ ID NO: 15.

[0310] In one aspect, the hinge or spacer comprises an IgG4 hinge. For example, in one embodiment, the hinge or spacer comprises the amino acid sequence [ka] In some embodiments, the hinge or spacer comprises a hinge of [ka] and a hinge encoded by the nucleotide sequence of

[0311] In one aspect, the hinge or spacer comprises an IgD hinge. For example, in one embodiment, the hinge or spacer comprises the amino acid sequence [ka] In some embodiments, the hinge or spacer comprises a hinge of [ka] and a hinge encoded by the nucleotide sequence of

[0312] In one aspect, the transmembrane domain may be recombinant, in which case it comprises primarily hydrophobic residues such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan, and valine may be found at each end of the recombinant transmembrane domain.

[0313] Optionally, a short oligo- or polypeptide linker, 2-10 amino acids in length, can form the linkage between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet provides a particularly suitable linker. For example, in one aspect, the linker comprises [ka] In some embodiments, the linker comprises the amino acid sequence [ka] It is encoded by the nucleotide sequence

[0314] In one aspect, the hinge or spacer comprises a KIR2DS2 hinge.

[0315] Cytoplasmic domain The cytoplasmic domain or region of a CAR comprises an intracellular signaling domain. The intracellular signaling domain is generally responsible for activating at least one of the normal effector functions of the immune cell into which the CAR is introduced. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. The term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the complete chain, so long as it transmits the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0316] Examples of intracellular signaling domains for use in the CARs of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that work together to initiate signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences and any recombinant sequences that have the same functional capacity.

[0317] It is known that signals generated via the TCR alone are insufficient for full activation of T cells, and that secondary and / or costimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains).

[0318] The primary signaling domain controls the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0319] Examples of ITAMs comprising primary intracellular signaling domains that are particularly useful in the present invention include those of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In some embodiments, a CAR of the invention comprises an intracellular signaling domain, e.g., the primary signaling domain of CD3 zeta.

[0320] In some embodiments, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain, that has altered (e.g., increased or decreased) activity compared to the native ITAM domain. In some embodiments, the primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In some embodiments, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

[0321] Further examples of primary intracellular signaling domain-containing molecules that are particularly useful in the present invention include those of DAP10, DAP12 and CD32.

[0322] The intracellular signaling domain of the CAR may comprise a CD3 zeta signaling domain alone, or may be combined with any other desired intracellular signaling domain useful for the CAR in the context of the present invention. For example, the intracellular signaling domain of the CAR may comprise a CD3 zeta chain portion and a costimulatory signaling domain. The costimulatory signaling domain refers to the portion of the CAR that comprises the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is necessary for the efficient response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. For example, CD27 costimulation has been shown to enhance the proliferation, effector function, and survival of human CART cells in vitro and to enhance human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3):696-706).Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, and CD29. , ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), NKG2D, CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), P Contains SGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp and CD19a.

[0323] The intracellular signaling sequences within the cytoplasmic portion of the CAR of the present invention can be linked to each other randomly or in a specific order. Optionally, a short oligo- or polypeptide linker, e.g., 2 to 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length, can form the linkage between the intracellular signaling sequences. In some embodiments, a glycine-serine doublet can be used as a suitable linker. In some embodiments, a single amino acid, e.g., alanine, glycine, can be used as a suitable linker.

[0324] In one aspect, the intracellular signaling domain is designed to comprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains. In some embodiments, the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains are separated by a linker molecule, e.g., a linker molecule described herein. In some embodiments, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0325] In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3 zeta and the signaling domain of CD28. In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3 zeta and the signaling domain of 4-1BB. In one aspect, the signaling domain of 4-1BB is the signaling domain of SEQ ID NO: 16. In one aspect, the signaling domain of CD3 zeta is the signaling domain of SEQ ID NO: 17.

[0326] In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3 zeta and the signaling domain of CD27. In one aspect, the signaling domain of CD27 is [ka] In one aspect, the signaling domain of CD27 comprises the amino acid sequence [ka] It is encoded by the nucleic acid sequence of

[0327] In some aspects, a CAR-expressing cell described herein can further comprise a second CAR, e.g., a second CAR that comprises a different antigen binding domain, e.g., to the same target (CD19) or to a different target (e.g., CD123 or mesothelin). In some embodiments, when a CAR-expressing cell comprises two or more different CARs, the antigen binding domains of the different CARs can be such that the antigen binding domains do not interact with each other. For example, a cell expressing a first and second CAR can have the antigen binding domain of the first CAR, e.g., as a fragment, e.g., an scFv, which does not bind to the antigen binding domain of the second CAR, e.g., the antigen binding domain of the second CAR is a VHH.

[0328] In other aspects, the CAR-expressing cells described herein can further express other agents, e.g., agents that enhance the activity of the CAR-expressing cells. For example, in some embodiments, the agent can be an agent that inhibits an inhibitory molecule. An inhibitory molecule, e.g., PD1, can, in some embodiments, reduce the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In some embodiments, the agent that inhibits an inhibitory molecule comprises, e.g., a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide of an inhibitory molecule such as, e.g., PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., as described herein, e.g., 41BB, CD27 or CD28) and / or a primary signaling domain). and a second polypeptide that is a CD3 zeta signaling domain described herein (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a CD3 zeta signaling domain described herein). PD1 is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75).Two ligands for PD1, PD-L1 and PD-L2, have been shown to downregulate T cell activation by binding to PD1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction between PD1 and PD-L1.

[0329] In some embodiments, an agent comprising the extracellular domain (ECD) of an inhibitory molecule, e.g., programmed cell death 1 (PD1), can be fused to a transmembrane domain and an intracellular signaling domain, such as 41BB and CD3 zeta (also referred to herein as a PD1 CAR). In some embodiments, the PD1 CAR improves T cell persistence when used in combination with a CD19 CAR described herein. In some embodiments, the CAR is a PD1 CAR comprising the extracellular domain of PD1 underlined in SEQ ID NO: 121. In some embodiments, the PD1 CAR comprises the amino acid sequence of SEQ ID NO: 121. [ka]

[0330] In one embodiment, the PD1 CAR comprises the amino acid sequence provided below (SEQ ID NO: 119). [ka]

[0331] In some embodiments, the agent comprises a nucleic acid sequence encoding a PD1 CAR, e.g., a PD1 CAR described herein. In some embodiments, the nucleic acid sequence for a PD1 CAR is shown below, with the PD1 ECD underlined below in SEQ ID NO: 120. [ka]

[0332] In another aspect, the present invention provides a population of CAR-expressing cells, e.g., CART cells. In some embodiments, the population of CAR-expressing cells comprises a mixture of cells expressing different CARs. For example, in some embodiments, the population of CAR-expressing cells can include a first cell expressing a CAR having an anti-CD19 binding domain described herein and a second cell expressing a CAR having a different anti-CD19 binding domain, e.g., an anti-CD19 binding domain described herein that is different from the anti-CD19 binding domain in the CAR expressed by the first cell. As another example, the population of CAR-expressing cells can include a first cell expressing a CAR comprising an anti-CD19 binding domain, e.g., as described herein, and a second cell expressing a CAR comprising an antigen binding domain that targets other than CD19 (e.g., CD123). In some embodiments, the population of CAR-expressing cells includes, e.g., a first cell expressing a CAR comprising a primary intracellular signaling domain and a second cell expressing a CAR comprising a secondary signaling domain.

[0333] In another aspect, the present invention provides a population of cells, wherein at least one cell in the population expresses a CAR having an anti-CD19 binding domain described herein, and a second cell expresses another agent, e.g., an agent that enhances the activity of the CAR-expressing cell. For example, in some embodiments, the agent can be an agent that inhibits an inhibitory molecule. The inhibitory molecule can, for example, in some embodiments, inhibit a CAR-expressing cell from mounting an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR beta. In some embodiments, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that transmits a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR beta, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described herein) and / or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein)). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a CD3 zeta signaling domain described herein).

[0334] Regulatable chimeric antigen receptors In some embodiments, regulatable CARs (RCARs) whose CAR activity can be controlled are desired to optimize the safety and efficacy of CAR therapy. There are many ways in which CAR activity can be controlled. For example, inducible apoptosis, such as using a caspase fused to a dimerization domain (see, e.g., Di Stasa et al., N Engl. J. Med. 2011 Nov. 3; 365(18):1673-1683), can be used as a safety switch in the CAR therapy of the present invention. In some embodiments, cells (e.g., T cells or NK cells) expressing the CAR of the present invention can further comprise an inducible apoptosis switch, in which a human caspase (e.g., caspase 9) or a modified version is fused to a modified version of the human FKB protein, which allows conditional dimerization. In the presence of a small molecule such as a rapalog (e.g., AP1903, AP20187), an inducible caspase (e.g., caspase 9) is activated, leading to rapid apoptosis and death of cells (e.g., T cells or NK cells) expressing the CAR of the invention. Examples of caspase-based inducible apoptosis switches (or one or more aspects of such switches) are described in, for example, US2004040047; US20110286980; US20140255360; WO1997031899; WO2014151960; WO2014164348; WO2014197638; WO2014197638, all of which are incorporated herein by reference.

[0335] In one aspect, an RCAR comprises a set of polypeptides, generally two in the simplest embodiments, in which the components of a standard CAR described herein, e.g., an antigen binding domain and an intracellular signaling domain, are located on separate polypeptides or members. In one embodiment, the set of polypeptides comprises a dimerization switch that can link the polypeptides to each other in the presence of a dimerization molecule, e.g., can link the antigen binding domain and the intracellular signaling domain. In one embodiment, the CAR of the invention utilizes a dimerization switch, such as that described in WO2014127261, incorporated herein by reference.

[0336] In one aspect, an RCAR comprises two polypeptides or members: 1) an intracellular signaling member comprising an intracellular signaling domain, e.g., a primary intracellular signaling domain described herein, and a first switch domain; and 2) an antigen binding member comprising an antigen binding domain that targets CD19 as described herein, e.g., as described herein, and a second switch domain. Optionally, the RCAR comprises a transmembrane domain described herein. In some embodiments, the transmembrane domain can be disposed on the intracellular signaling member, the antigen binding member, or both. (Unless otherwise specified, when members or elements of an RCAR are described herein, the order can be as shown, but other orders are included as well. In other words, in some embodiments, the order is as described herein, but in other embodiments, the order can be different. For example, the order of elements on one side of the transmembrane region can be different from the examples, e.g., the position of the switch domain relative to the intracellular signaling domain can be different, e.g., reversed.)

[0337] In some embodiments, the first and second switch domains can form an intracellular or extracellular dimerization switch. In some embodiments, the dimerization switch can be a homodimerization switch, e.g., when the first and second switch domains are the same, or a heterodimerization switch, e.g., when the first and second switch domains are different from one another.

[0338] In some embodiments, an RCAR can comprise a "multiple switch." A multiple switch can comprise a heterodimerization switch domain or a homodimerization switch domain. A multiple switch comprises a plurality of switch domains, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, independently of a first member, e.g., an antigen-binding member, and a second member, e.g., an intracellular signaling member. In some embodiments, a first member can comprise a plurality of first switch domains, e.g., FKBP-based switch domains, and a second member can comprise a plurality of second switch domains, e.g., FRB-based switch domains. In some embodiments, a first member can comprise a first and a second switch domain, e.g., an FKBP-based switch domain and an FRB-based switch domain, and a second member can comprise a first and a second switch domain, e.g., an FKBP-based switch domain and an FRB-based switch domain.

[0339] In some embodiments, the intracellular signaling member comprises one or more intracellular signaling domains, e.g., a primary intracellular signaling domain and one or more costimulatory signaling domains.

[0340] In some embodiments, the antigen binding member can comprise one or more intracellular signaling domains, e.g., one or more costimulatory signaling domains. In some embodiments, the antigen binding member comprises multiple, e.g., two or three, costimulatory signaling domains described herein, e.g., selected from 41BB, CD28, CD27, ICOS, and OX40, and in some embodiments, no primary intracellular signaling domain. In some embodiments, the antigen binding member comprises, from extracellular to intracellular, the following costimulatory signaling domains: 41BB-CD27; 41BB-CD27; CD27-41BB; 41BB-CD28; CD28-41BB; OX40-CD28; CD28-OX40; CD28-41BB; or 41BB-CD28. In such embodiments, the intracellular binding member comprises a CD3 zeta domain. In one such embodiment, the RCAR comprises: (1) an antigen binding member comprising an antigen binding domain, a transmembrane domain, and two costimulatory domains and a first switch domain; and (2) an intracellular signaling domain comprising a transmembrane domain or membrane tethering domain and at least one primary intracellular signaling domain and a second switch domain.

[0341] One embodiment provides an RCAR in which the antigen binding member is not tethered to the surface of the CAR cell. This allows cells having an intracellular signaling member to be conveniently paired with one or more antigen binding domains without transforming the cell with a sequence encoding the antigen binding member. In such embodiments, the RCAR comprises: 1) an intracellular signaling member comprising a first switch domain, a transmembrane domain, and an intracellular signaling domain, e.g., a primary intracellular signaling domain and a first switch domain; and 2) an antigen binding member comprising an antigen binding domain and a second switch domain, wherein the antigen binding member does not comprise a transmembrane domain or a membrane tethering domain, and optionally does not comprise an intracellular signaling domain. In some embodiments, the RCAR can further comprise: 3) a second antigen binding domain, e.g., a second antigen binding domain, that binds to a different antigen than that bound by the antigen binding domain; and a second antigen binding member comprising a second switch domain.

[0342] Also provided is an RCAR in which the antigen binding member comprises bispecific activation and targeting capabilities. In this embodiment, the antigen binding member can comprise multiple, for example, 2, 3, 4, or 5, antigen binding domains, for example, scFvs, where each antigen binding domain binds to a target antigen, for example, different antigens, or the same antigen, for example, the same or different epitopes on the same antigen. In some embodiments, the multiple antigen binding domains are tandem, and optionally, a linker or hinge region is disposed between each of the antigen binding domains. Suitable linkers and hinge regions are described herein.

[0343]

[0010] One embodiment provides an RCAR having a configuration that allows proliferation switching. In this embodiment, the RCAR comprises: 1) an intracellular signaling member, optionally comprising a transmembrane domain or membrane tethering domain; e.g., one or more costimulatory signaling domains and a switch domain selected from 41BB, CD28, CD27, ICOS, and OX40; and 2) an antigen binding member comprising an antigen binding domain, a transmembrane domain, and a primary intracellular signaling domain, e.g., a CD3 zeta domain, wherein the antigen binding member does not comprise a switch domain or does not comprise a switch domain that dimerizes with a switch domain on the intracellular signaling member. In some embodiments, the antigen binding member does not comprise a costimulatory signaling domain. In some embodiments, the intracellular signaling member comprises a switch domain from a homodimerization switch. In some embodiments, the intracellular signaling member comprises a first switch domain of a heterodimerization switch, and the RCAR comprises a second intracellular signaling member comprising a second switch domain of the heterodimerization switch. In such embodiments, the second intracellular signaling member comprises the same intracellular signaling domain as the intracellular signaling member. In some embodiments, the dimerization switch is intracellular. In some embodiments, the dimerization switch is extracellular.

[0344] In any of the RCAR configurations described herein, the first and second switch domains comprise an FKBP-FRB-based switch as described herein.

[0345] Also provided herein are cells comprising the RCARs described herein. Any cells engineered to express RCAR can be used as RCARX cells. In some embodiments, RCARX cells are T cells and are referred to as RCAR cells. In some embodiments, RCARX cells are NK cells and are referred to as RCARN cells.

[0346] Nucleic acids and vectors comprising RCAR-encoding sequences are also provided herein. Sequences encoding various elements of RCAR can be located on the same nucleic acid molecule, e.g., the same plasmid or vector, e.g., a viral vector, e.g., a lentiviral vector. In some embodiments, (i) a sequence encoding an antigen-binding member and (ii) a sequence encoding an intracellular signaling member can be present on the same nucleic acid, e.g., vector. Production of the corresponding proteins can be achieved, for example, by using separate promoters or by using bicistronic transcription products (which can result in the production of two proteins by cleavage of a single translation product or by translation of two separate protein products). In some embodiments, a sequence encoding a cleavable peptide, e.g., a P2A or F2A sequence, is located between (i) and (ii). In some embodiments, a sequence encoding an IRES, e.g., an EMCV or EV71 IRES, is located between (i) and (ii). In these embodiments, (i) and (ii) are transcribed as a single RNA. In one embodiment, a first promoter is operably linked to (i) and a second promoter is operably linked to (ii), such that (i) and (ii) are transcribed as separate mRNAs.

[0347] Alternatively, the sequences encoding the various elements of the RCAR can be located on different nucleic acid molecules, e.g., different plasmids or vectors, e.g., viral vectors, e.g., lentiviral vectors. For example, (i) the sequence encoding the antigen-binding member can be present on a first nucleic acid, e.g., a first vector, and (ii) the sequence encoding the intracellular signaling member can be present on a second nucleic acid, e.g., a second vector.

[0348] Dimerization switch Dimerization switches can be non-covalent or covalent. In non-covalent dimerization switches, the dimerization molecule promotes non-covalent interactions between the switch domains. In covalent dimerization switches, the dimerization molecule promotes covalent interactions between the switch domains.

[0349] In some embodiments, the RCAR comprises an FKBP / FRAP or FKBP / FRB-based dimerization switch. FKBP12 (FKBP or FK506-binding protein) is an abundant cytoplasmic protein that serves as the initial intracellular target of the natural product immunosuppressant, rapamycin. Rapamycin binds to FKBP and the large PI3K homolog FRAP (RAFT, mTOR). FRB is a 93-amino acid portion of FRAP that is sufficient for binding of the FKBP-rapamycin complex (Chen, J., Zheng, XF, Brown, EJ & Schreiber, SL (1995) Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci USA 92: 4947-51).

[0350] In some embodiments, an FKBP / FRAP, e.g., an FKBP / FRB-based switch, can be used as the dimerization molecule, e.g., rapamycin or a rapamycin analog. The amino acid sequence of FKBP is as follows: [ka]

[0351] In certain embodiments, an FKBP switch domain can comprise a fragment of FKBP, for example, the underlined portion of SEQ ID NO:37, below, that has the ability to bind to FRB, or a fragment or analog thereof, in the presence of rapamycin or a rapalog. [ka]

[0352] The amino acid sequence of FRB is as follows: [ka]

[0353] An "FKBP / FRAP, e.g., FKBP / FRB-based switch," as that term is used herein, refers to an FKBP or its FRAP-based switch that has the ability to bind to FRB, or a fragment or analog thereof, in the presence of rapamycin or a rapalog, e.g., RAD001, and that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue from, the FKBP sequence of SEQ ID NO: 122 or 123. and a second switch domain comprising FRB or a fragment or analog thereof, which has the ability to bind to FRB or a fragment or analog thereof in the presence of rapamycin or a rapalog and which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue from, the FRB sequence of SEQ ID NO: 124. In one embodiment, an RCAR described herein comprises one switch domain comprising the amino acid residues disclosed in SEQ ID NO: 122 (or SEQ ID NO: 123) and one switch domain comprising the amino acid residues disclosed in SEQ ID NO: 124.

[0354] In some embodiments, the FKBP / FRB dimerization switch comprises a modified FRB switch domain, e.g., a modified FRB switch domain, that has altered, e.g., enhanced, complex formation between an FKBP-based switch domain and a dimerization molecule, e.g., rapamycin or a rapalog, e.g., RAD001. In some embodiments, the modified FRB switch domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations selected from mutations at amino acid positions L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105, and F2108, where the wild-type amino acid is mutated to another naturally occurring amino acid. In some embodiments, the mutant FRB comprises a mutation at E2032, where E2032 is mutated to phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I), e.g., SEQ ID NO: 125, or leucine (E2032L), e.g., SEQ ID NO: 126. In some embodiments, the mutant FRB comprises a mutation at T2098, where T2098 is mutated to phenylalanine (T2098F) or leucine (T2098L), e.g., SEQ ID NO: 127. In some embodiments, the mutant FRB comprises mutations at E2032 and T2098, where E2032 is mutated to any amino acid and T2098 is mutated to any amino acid, e.g., SEQ ID NO: 128. In some embodiments, the mutant FRB comprises an E2032I and a T2098L mutation, e.g., SEQ ID NO: 129. In some embodiments, the mutant FRB comprises an E2032L and a T2098L mutation, e.g., SEQ ID NO: 130.

[0355] [Table 1]

[0356] Other suitable dimerization switches include GyrB-GyrB-based dimerization switches, gibberellin-based dimerization switches, tag / binder dimerization switches, and halotag / snaptag dimerization switches. Following the guidance provided herein, such switches and associated dimerization molecules will be apparent to those of skill in the art.

[0357] dimerization molecule Association between the switch domains is promoted by a dimerization molecule. In the presence of a dimerization molecule, interaction or association between the switch domains allows signaling between a polypeptide bound to, e.g., fused to, a first switch domain and a polypeptide bound to, e.g., fused to, a second switch domain. In the presence of a non-limiting level of a dimerization molecule, signaling is increased by, e.g., 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 5-fold, 10-fold, 50-fold, or 100-fold, as measured, e.g., in a system described herein.

[0358] Rapamycin and rapamycin analogs (sometimes referred to as rapalogs), e.g., RAD001, can be used as dimerization molecules in the FKBP / FRB-based dimerization switches described herein. In some embodiments, the dimerization molecule can be selected from rapamycin (sirolimus), RAD001 (everolimus), zotarolimus, temsirolimus, AP-23573 (ridaforolimus), biolimus, and AP21967. Additional rapamycin analogs suitable for use with FKBP / FRB-based dimerization switches are further described in the section entitled "Combination Therapies" or in the section entitled "Representative mTOR Inhibitors."

[0359] split CAR In some embodiments, the CAR-expressing cells use split CARs. Split CAR techniques are described in further detail in publications WO2014 / 055442 and WO2014 / 055657. Briefly, the split CAR system comprises a cell that expresses a first CAR having a first antigen binding domain and a costimulatory domain (e.g., 41BB), and the cell also expresses a second CAR having a second antigen binding domain and an intracellular signaling domain (e.g., CD3 zeta). When the cell encounters the first antigen, the costimulatory domain is activated, causing the cell to proliferate. When the cell encounters the second antigen, the intracellular signaling domain is activated, initiating cell killing activity. Therefore, the CAR-expressing cells are only fully activated in the presence of both antigens.

[0360] RNA transfection Disclosed herein are methods for producing in vitro transcribed RNA CARs. The present invention also includes CAR-encoding RNA constructs that can be directly transfected into cells. Methods for producing mRNA for use in transfection involve in vitro transcription (IVT) of a template using specially designed primers to produce a construct containing 3' and 5' untranslated sequences ("UTRs"), a 5' cap and / or internal ribosome entry site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length (SEQ ID NO: 118), followed by polyA addition. RNAs produced in this manner can efficiently transfect cells of different species. In one aspect, the template contains the sequence for the CAR.

[0361] In one aspect, the anti-CD19 CAR is encoded by messenger RNA (mRNA). In one aspect, the mRNA encoding the anti-CD19 CAR is introduced into an immune effector cell, e.g., a T cell or an NK cell, for production of a CAR-expressing cell, e.g., a CART cell or a CAR NK cell.

[0362] In some embodiments, in vitro transcribed RNA CAR can be introduced into cells in the form of transient transfection. RNA is produced by in vitro transcription using a template produced by polymerase chain reaction (PCR). Target DNA from any source can be directly converted into a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable source of DNA. The desired template for in vitro transcription is the CAR of the present invention. For example, the template for RNA CAR comprises an extracellular region including a single-chain variable domain of an anti-tumor antibody; a hinge region, a transmembrane domain (for example, the transmembrane domain of CD8a); and a cytoplasmic region including an intracellular signaling domain, for example, including the signaling domain of CD3 zeta and the signaling domain of 4-1BB.

[0363] In some embodiments, the DNA used for PCR contains an open reading frame. The DNA can be derived from a naturally occurring DNA sequence from the genome of an organism. In some embodiments, the nucleic acid can include some or all of the 5' and / or 3' untranslated regions (UTRs). The nucleic acid can include exons and introns. In some embodiments, the DNA used for PCR is a human nucleic acid sequence. In other embodiments, the DNA used for PCR is a human nucleic acid sequence including the 5' UTR and 3' UTR. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in naturally occurring organisms. An exemplary artificial DNA sequence is one that contains portions of genes ligated together to encode an open reading frame that encodes a fusion protein. The portions of DNA ligated together can be from a single organism or from more than one organism.

[0364] PCR is used to generate templates for in vitro transcription of mRNA for use in transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have a region that is substantially complementary to a region of DNA to be used as a template in PCR. As used herein, "substantially complementary" refers to a nucleotide sequence in which most or all of the bases in the primer sequence are complementary, or in which one or more bases are non-complementary or mismatched. A substantially complementary sequence can anneal or hybridize with the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify a portion of a nucleic acid that is normally transcribed in cells (open reading frame), including the 5' UTR and 3' UTR. Primers can also be designed to amplify a portion of a nucleic acid encoding a specific domain of interest. In one embodiment, primers are designed to amplify the coding region of a human cDNA, including all or part of the 5' UTR and 3' UTR. Primers useful for PCR can be produced by synthetic methods well known in the art. A "forward primer" is a primer that contains a region of nucleotides that are substantially complementary to nucleotides on a DNA template that are upstream of the DNA sequence to be amplified. "Upstream" refers to a position 5' to the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that contains a region of nucleotides that are substantially complementary to a double-stranded DNA template that is downstream of the DNA sequence to be amplified. "Downstream" refers to a position 3' to the DNA sequence to be amplified relative to the coding strand.

[0365] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. Reagents and polymerases are available from a number of commercial sources.

[0366] Chemical structures capable of promoting stability and / or translation efficiency may also be used. The RNA preferably has 5' and 3' UTRs. In some embodiments, the 5' UTR is 1 to 3,000 nucleotides long. The length of the 5' and 3' UTR sequences to be added to the coding region can be varied using different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTR. Using this method, those skilled in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0367] The 5' and 3' UTRs can be naturally occurring endogenous 5' and 3' UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating UTR sequences into forward and reverse primers or other modifications to the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in 3' UTR sequences can reduce mRNA stability. Therefore, 3' UTRs can be selected or designed to increase the stability of transcribed RNA based on the properties of UTRs, which are well known in the art.

[0368] In some embodiments, the 5' UTR can contain the Kozak sequence of the endogenous nucleic acid. Alternatively, when a non-endogenous 5' UTR is added to the nucleic acid of interest by PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. While the Kozak sequence can increase the translation efficiency of some RNA transcripts, it is not believed to be necessary for all RNAs to enable efficient translation. The necessity of the Kozak sequence for many mRNAs is known in the art. In other embodiments, the 5' UTR can be the 5' UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' or 5' UTR to prevent exonuclease degradation of the mRNA.

[0369] To enable RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter must be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In a preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3, and SP6 promoters are known in the art.

[0370] In a preferred embodiment, the mRNA has both a 5'-end cap and a 3'-poly(A) tail, which determine ribosome binding, translation initiation, and stability in cells. In a circular DNA template, such as a plasmid DNA, RNA polymerase produces long, chain-like products that are unsuitable for expression in eukaryotic cells. Transcription of linearized plasmid DNA at the end of the 3' UTR results in mRNA of normal size, which is not effective for eukaryotic transfection even after post-transcriptional polyadenylation.

[0371] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003)).

[0372] The traditional method for incorporating a polyA / T stretch into a DNA template is molecular cloning. However, integration of a polyA / T sequence into plasmid DNA can destabilize the plasmid, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other abnormalities. This makes the cloning method not only tedious and time-consuming, but also often unreliable. This is why a method that allows the construction of a DNA template with a polyA / T 3' stretch without cloning is highly desirable.

[0373] A poly(A) / T stretch can be integrated into a DNA template (e.g., a plasmid) by molecular cloning. Alternatively, a poly(A) / T segment of a transcribable DNA template can be generated during PCR using a reverse primer containing a poly(T) tail, such as a 100T tail (SEQ ID NO: 110) (which can range in size from 50 to 5000T (SEQ ID NO: 111)), or post-PCR by other methods, including, but not limited to, DNA ligation or in vitro recombination. The poly(A) tail also confers stability to RNA and reduces its degradation. Generally, the length of the poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100 to 5000 adenosines (SEQ ID NO: 112).

[0374] The poly(A) tail of RNA can be further extended after in vitro transcription by using a poly(A) polymerase, such as E. coli poly(A) polymerase (E-PAP). In some embodiments, extending the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides (SEQ ID NO: 113) results in an approximately two-fold increase in RNA translation efficiency. Furthermore, attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachments can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase RNA stability.

[0375] A 5' cap also provides stability to an RNA molecule. In preferred embodiments, the RNA produced by the methods disclosed herein includes a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7: 1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0376] The RNA produced by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and promotes translation initiation. Any solute suitable for cell electroporation may be included, including factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and detergents.

[0377] RNA can be integrated into target cells using any of a number of different methods, including, but not limited to, commercially available methods such as electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (Biorad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0378] Non-viral delivery methods In some aspects, non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein to a cell or tissue or subject.

[0379] In some embodiments, non-viral methods involve the use of transposons (also called transposable elements). In some embodiments, a transposon is a piece of DNA that can insert itself into a certain location in a genome, for example, a piece of DNA that can self-replicate and insert its copy into a genome, or a piece of DNA that can be excised from a long nucleic acid and inserted into another location in the genome. For example, a transposon is a DNA sequence consisting of an inverted repeat adjacent to a gene for transposition.

[0380] Representative methods for nucleic acid delivery using transposons include the Sleeping Beauty transposon system (SBTS) and the piggyBac (PB) transposon system. See, e.g., Aronovich et al. Hum. Mol. Genet. 20.R1(2011):R14-20; Singh et al. Cancer Res. 15(2008):2961-2971; Huang et al. Mol. Ther. 16(2008):580-589; Grabundzija et al. Mol. Ther. 18(2010):1200-1209; Kebriaei et al. Blood. 122.21(2013):166; Williams. Molecular Therapy 16.9(2008):1515-16; Bell et al. Nat. Protoc. 2.12(2007):3153-65; and Ding et al. Cell. 122.3(2005):473-83, all of which are incorporated herein by reference.

[0381] SBTS contains two components: 1) a transposon containing a transgene and 2) a source of transposase enzyme. The transposase can transpose the transposon from a carrier plasmid (or other donor DNA) into target DNA, such as a host cell chromosome / genome. For example, the transposase binds to the carrier plasmid / donor DNA, excises the transposon (containing the transgene) from the plasmid, and introduces it into the genome of the host cell. See, e.g., Aronovich et al. supra.

[0382] Representative transposons include pT2-based transposons. See, for example, Grabundzija et al. Nucleic Acids Res. 41.3(2013):1829-47; and Singh et al. Cancer Res. 68.8(2008):2961-2971, all of which are incorporated herein by reference. Representative transposases include Tc1 / mariner-type transposases, such as SB10 transposase or SB11 transposase (e.g., hyperactive transposases that can be expressed from a cytomegalovirus promoter). See, for example, Aronovich et al.; Kebriaei et al.; and Grabundzija et al., all of which are incorporated herein by reference.

[0383] The use of SBTS allows for efficient integration and expression of a transgene, e.g., a nucleic acid encoding a CAR described herein. For example, provided herein are methods for producing cells, e.g., T cells or NK cells, that stably express a CAR described herein using a transposon system such as SBTS.

[0384] In some embodiments, one or more nucleic acids, e.g., plasmids, comprising an SBTS element are delivered to cells (e.g., T cells or NK cells) by the methods described herein. For example, the nucleic acid is delivered by standard methods for nucleic acid (e.g., plasmid DNA) delivery, e.g., methods described herein, e.g., electroporation, transfection, or lipofection. In some embodiments, the nucleic acid comprises a transposon comprising a transgene, e.g., a nucleic acid encoding a CAR described herein. In some embodiments, the nucleic acid comprises a transposon comprising a transgene (e.g., a nucleic acid encoding a CAR described herein) and a nucleic acid sequence encoding a transposase enzyme. In other embodiments, a system involving two nucleic acids is provided, e.g., a dual-plasmid system, in which a first plasmid comprises a transposon comprising a transgene and a second plasmid comprises a nucleic acid sequence encoding a transposase enzyme. For example, the first and second nucleic acids are co-delivered to the host cell.

[0385] In some embodiments, cells, e.g., T cells or NK cells, that express a CAR described herein are produced by using a combination of gene insertion using SBTS and gene editing using nucleases (e.g., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR / Cas systems, or engineered meganuclease redesigned homing endonucleases).

[0386] In some embodiments, the use of non-viral delivery methods allows for the reprogramming of cells, such as T cells or NK cells, and the direct infusion of the cells into a subject. The advantages of non-viral vectors include, but are not limited to, the ease and relatively low cost of producing sufficient quantities to meet the patient population, stability during storage, and lack of immunogenicity.

[0387] Nucleic acid construct encoding a CAR The present invention also provides nucleic acid molecules encoding one or more of the CAR constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct.

[0388] Accordingly, in one aspect, the invention relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-CD19 binding domain (e.g., a humanized anti-CD19 binding domain), a transmembrane domain, and a stimulatory domain, e.g., a costimulatory signaling domain and / or a primary signaling domain, e.g., an intracellular signaling domain comprising a zeta chain. In one embodiment, the anti-CD19 binding domain is an anti-CD19 binding domain described herein, e.g., an anti-CD19 binding domain comprising a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:59, or a sequence having 95-99% identity thereto. In some embodiments, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 15, or a sequence having 95-99% identity thereto. In some embodiments, the anti-CD19 binding domain is connected to the transmembrane domain by a hinge region, e.g., a hinge described herein. In some embodiments, the hinge region comprises SEQ ID NO: 14, or SEQ ID NO: 45, or SEQ ID NO: 47, or SEQ ID NO: 49, or a sequence having 95-99% identity thereto. In some embodiments, the isolated nucleic acid molecule further comprises a sequence encoding a costimulatory domain. In some embodiments, the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). In some embodiments, the costimulatory domain comprises the sequence of SEQ ID NO: 16, or a sequence having 95-99% identity thereto. In some embodiments, the intracellular signaling domain comprises the functional signaling domain of 4-1BB and the functional signaling domain of CD3 zeta.In some embodiments, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 or SEQ ID NO: 51, or a sequence with 95-99% identity thereof, and the sequence of SEQ ID NO: 17 or SEQ ID NO: 43, or a sequence with 95-99% identity thereof, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

[0389] In another aspect, the invention pertains to an isolated CAR-encoding nucleic acid molecule construct comprising a leader sequence of SEQ ID NO: 13, an scFv domain having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 59 (or a sequence with 95 to 99% identity thereof), a hinge region of SEQ ID NO: 14 or SEQ ...

Claims

1. 1. A composition comprising cells (e.g., a population of cells) expressing a CAR molecule that binds CD19 ("CAR19-expressing cells"), for use in combination with one or more kinase inhibitors in the treatment of a mammal having a disease associated with CD19 expression, wherein the kinase inhibitor is selected from a Bruton's tyrosine kinase (BTK) inhibitor, a cyclin-dependent kinase 4 (CDK4) inhibitor, an mTOR inhibitor, or a mitogen-activated protein kinase-interacting kinase (MNK) inhibitor.

2. A method for treating a mammal having a disease associated with expression of CD19, comprising administering to the mammal an effective amount of cells (e.g., a population of cells) expressing a CAR molecule that binds to CD19 (CAR19-expressing cells), in combination with one or more kinase inhibitors selected from a Bruton's tyrosine kinase (BTK) inhibitor, a cyclin-dependent kinase 4 (CDK4) inhibitor, an mTOR inhibitor, or a mitogen-activated protein kinase-interacting kinase (MNK) inhibitor.

3. 3. The use or method of claim 1 or 2, wherein the kinase inhibitor and the CAR19-expressing cells are administered to the mammal as a first line of treatment.

4. 3. The use or method of claim 1 or 2, wherein the CAR19-expressing cells are administered to the mammal after administration of a kinase inhibitor.

5. (i) administering the CAR19-expressing cells after cessation of administration of the kinase inhibitor; or (ii) administration of the kinase inhibitor is initiated prior to administration of the CAR19-expressing cells, and the CAR19-expressing cells are administered in combination with the ongoing administration of the kinase inhibitor; 5. The use or method of claim 4.

6. 5. The use or method of any of claims 1 to 4, wherein the mammal is or is determined to be a complete or partial responder to a BTK inhibitor (e.g., ibrutinib) or a complete or partial responder to CAR19-expressing cells.

7. 7. The use or method of any of claims 1 to 6, wherein the BTK inhibitor is selected from ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13.

8. 7. The use or method of any one of claims 1 to 6, wherein the CDK4 inhibitor is selected from palbociclib, aloisine A, flavopiridol, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone, crizotinib (PF-02341066, P276-00, RAF265, indisulam roscovitine, dinaciclib, BMS 387032, MLN8054, AG-024322, AT7519, AZD5438, BMS908662, or ribociclib.

9. 7. The use or method of any of claims 1 to 6, wherein the mTOR inhibitor is selected from rapamycin, everolimus, temsirolimus, ridaforolimus, semapimod, AZD8055, PF04691502, SF1126, XL765 or a rapamycin analogue such as OSI-027.

10. 7. The use or method of any of claims 1 to 6, wherein the MNK inhibitor is selected from CGP052088, CGP57380, cercosporamide, ETC-1780445-2 or 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine.

11. 11. The use or method of any of claims 1 to 10, wherein the kinase inhibitor is ibrutinib and the ibrutinib is at a daily dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg or 600 mg.

12. The use or method of any of claims 1 to 11, wherein the cell expresses a CAR molecule comprising a CD19-binding domain, a transmembrane domain, and an intracellular signaling domain.

13. 13. The use or method of claim 12, wherein the intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

14. 14. The use or method of claim 12 or 13, wherein the CAR molecule comprises an anti-CD19 binding domain comprising light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), light chain complementarity determining region 3 (LC CDR3), heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3) of the anti-CD19 binding domain.

15. 15. The use or method of any of claims 12 to 14, wherein the anti-CD19 binding domain comprises a murine light chain variable region of Table 7, a murine heavy chain variable region of Table 7, or both.

16. The use or method of any of claims 12 to 15, wherein the anti-CD19 binding domain comprises a LC CDR1 of SEQ ID NO:5, a LC CDR2 of SEQ ID NO:26 and a LC CDR3 of SEQ ID NO:

27.

17. The use or method of any of claims 12 to 16, wherein the anti-CD19 binding domain comprises a HC CDR1 of SEQ ID NO: 19, a LC CDR2 of any of SEQ ID NOs: 20-23 and a HC CDR3 of SEQ ID NO:

24.

18. The use or method of any of claims 12 to 17, wherein the anti-CD19 binding domain comprises the sequence of SEQ ID NO: 59 or a sequence which is 95-99% identical thereto.

19. The use or method of any of claims 12 to 14, 16 or 17, wherein the anti-CD19 binding domain is a humanized anti-CD19 binding domain.

20. 20. The use or method of claim 19, wherein the humanized anti-CD19 binding domain comprises a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 or a sequence with 95-99% identity thereto.

21. 21. The use or method of claim 19 or 20, wherein the humanized anti-CD19 binding domain is an scFv comprising a light chain variable region linked to a heavy chain variable region via a linker, for example, the linker comprises the sequence of SEQ ID NO:

53.

22. 22. The use or method of any preceding claim, wherein the CAR molecule comprises a transmembrane domain of a protein selected from the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154.

23. 23. The use or method of claim 22, wherein the transmembrane domain comprises the sequence of SEQ ID NO:

15.

24. 13. The use or method of claim 12, wherein the anti-CD19 binding domain is connected to the transmembrane domain by a hinge region, for example, wherein the hinge region comprises the sequence of SEQ ID NO: 14 or SEQ ID NO:

45.

25. 25. The use or method of any of claims 1 to 24, wherein the CAR molecule comprises a costimulatory domain, e.g., wherein the costimulatory domain comprises a functional signaling domain of a protein selected from OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18) or 4-1BB (CD137), e.g., wherein the costimulatory domain comprises the sequence of SEQ ID NO: 16 or SEQ ID NO:

51.

26. The use or method of any of claims 1 to 25, wherein the CAR molecule comprises an intracellular signaling domain, for example, the intracellular signaling domain comprises a functional signaling domain of 4-1BB, a functional signaling domain of CD3 zeta, or both, or the intracellular signaling domain comprises a sequence of a functional signaling domain of CD27, CD3 zeta, or both.

27. 27. The use or method of claim 26, wherein the intracellular signaling domain comprises the sequence of SEQ ID NO: 16, the sequence of SEQ ID NO: 17 or both; wherein the intracellular signaling domain comprises the sequence of SEQ ID NO: 16, the sequence of SEQ ID NO: 43 or both; wherein the intracellular signaling domain comprises the sequence of SEQ ID NO: 51, the sequence of SEQ ID NO: 17 or both; or wherein the intracellular signaling domain comprises the sequence of SEQ ID NO: 51, the sequence of SEQ ID NO: 43 or both.

28. 28. The use or method of any preceding claim, wherein the CAR molecule further comprises a leader sequence, for example, wherein the leader sequence comprises the amino acid sequence of SEQ ID NO:

13.

29. 29. The use or method of any of claims 1 to 28, wherein the CAR molecule comprises the amino acid sequence of SEQ ID NO:58, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:

42.

30. 30. The use or method of any of claims 1 to 29, used in combination with an agent that inhibits an immune inhibitory molecule selected from PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta.

31. The composition is 1 to 5 × 10 ８ The use or method of any of claims 1 to 30, comprising a CAR-expressing cell.

32. The use or method according to any one of claims 1 to 31, wherein the disease associated with CD19 expression is cancer.

33. 33. The use or method of any of claims 1 to 32, wherein the disease associated with CD19 expression is a blood cancer, such as a blood cancer selected from leukemia or lymphoma.

34. Cancers include chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoid leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, and Burkitt's lymphoma. , diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, follicular lymphoma, childhood follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (mucosa-associated lymphoid tissue type extranodal marginal zone lymphoma), marginal zone lymphoma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, 33. The use or method of claim 32, wherein the tumor is selected from plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's hypergammaglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, lymphoplasmacytic lymphoma, heavy chain disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, nodular marginal zone lymphoma, childhood nodular marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma or unclassifiable lymphoma.

35. 33. The use or method of claim 32, wherein the cancer is selected from MCL, CLL, ALL, Hodgkin's lymphoma or multiple myeloma.

36. The use or method according to any one of claims 1 to 35, in combination with a cytokine.

37. 37. The use or method of claim 36, wherein the cytokine is IL-7, IL-15 or IL-21.

38. 38. The use or method of any preceding claim, wherein the CAR is a regulatable CAR (RCAR).

39. RCAR an intracellular signaling member comprising an intracellular signaling domain and a first switch domain; an antigen binding member comprising an antigen binding domain that binds to CD19 and a second switch domain; and Transmembrane domain 39. The use or method of claim 38, comprising:

40. 40. The use or method of any preceding claim, wherein the mammal has or is identified as having a BTK mutation.

41. 41. The use or method of any of claims 1 to 40, wherein the disease associated with expression of CD19 is a hematological cancer and resistance to kinase inhibitors, cells expressing a CAR molecule in a mammal, or both, is delayed or reduced.

42. 42. The use or method according to any one of claims 1 to 41, wherein the disease associated with expression of CD19 is a blood cancer, and the remission of the blood cancer is prolonged or the recurrence of the blood cancer is delayed.

43. 3. The use or method of claim 1 or 2, wherein the CAR19-expressing cells are administered in combination with a second kinase inhibitor, wherein the second kinase inhibitor is other than ibrutinib when the mammal is, or has been identified as, a non-responder or relapser to ibrutinib.

44. 44. The use or method of claim 43, wherein the second kinase inhibitor is selected from one or more of GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774 or LFM-A13 or combinations thereof.

45. 45. The use or method of any of claims 1-44, wherein the mammal is (or is identified as) a partial responder to the kinase inhibitor, and the CAR19-expressing cells are administered to the mammal, alone or in combination with the BTK inhibitor, for the duration of the partial response.

46. 3. The use or method of claim 1 or 2, wherein the mammal is (or is identified as) a non-responder who has progressive or stable disease following treatment with ibrutinib, and wherein the mammal is administered CAR19-expressing cells, alone or in combination with a second BTK inhibitor, during the period of progressive or stable disease, wherein the second kinase inhibitor is other than ibrutinib.

47. 47. The use or method of any preceding claim, wherein the kinase inhibitor is ibrutinib and the ibrutinib is formulated for administration in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles, for example wherein the cycle length is 21 or 28 days.

48. 48. The use or method of any of claims 1 to 47, comprising performing a lymphocyte infusion with at least one CD19 CAR-expressing cell.

49. 49. The use or method of any preceding claim, wherein the cells and the kinase inhibitor are formulated for simultaneous administration.

50. 50. The use or method of any preceding claim, wherein the cells and the kinase inhibitor are formulated for sequential delivery.

51. 51. The use or method of any preceding claim, wherein the mammal has undergone lymphodepletion.

52. 52. The use or method of claim 51, wherein lymphodepletion comprises administration of one or more of melphalan, cytoxan, cyclophosphamide, and fludarabine.

53. 53. The use or method of any of claims 1-52, further comprising administering to the mammal a low, immune enhancing dose of an mTOR inhibitor.

54. 54. The use or method of claim 53, wherein the mTOR inhibitor is everolimus or rapamycin.

55. 1. A method of producing a CAR-expressing cell (e.g., a CAR-expressing immune effector cell) or population of cells, comprising: contacting the cell or population of cells with a BTK inhibitor; and A nucleic acid encoding the CAR molecule is introduced (e.g., transduced) into a cell or population of cells under conditions such that the CAR molecule is expressed. A method comprising:

56. 56. The method of claim 55, wherein the CAR molecule is a CAR molecule that binds to CD19.

57. 57. The method of claim 55 or 56, wherein the cells are T cells or NK cells, or the population of cells comprises T cells, NK cells, or both.

58. 58. The method of any of claims 55-57, wherein the cell or population of cells is contacted with the BTK inhibitor for 10-20 minutes, 20-30 minutes, 30-40 minutes, 40-60 minutes, or 60-120 minutes, and then most or all of the BTK inhibitor is removed from the cell or population of cells.

59. 59. The method of any of claims 55-58, wherein the BTK inhibitor is administered after the cell or population of cells is harvested or before stimulating the cell or population of cells.

60. 60. The method of any of claims 55-59, wherein the BTK inhibitor is selected from ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13.

61. 61. The method of any one of claims 55 to 60, wherein the population of cells also comprises cancer cells.

62. 62. The method of claim 61, wherein the BTK inhibitor inhibits BTK in cancer cells.

63. 63. The method of any of claims 55-62, further comprising depleting T regulatory cells (e.g., CD25+ cells) from the population of cells.

64. A reaction mixture comprising a population of immune effector cells, a BTK inhibitor, and a CAR molecule or a nucleic acid encoding a CAR molecule.

65. 65. The reaction mixture of claim 64, wherein one or more of the immune effector cells expresses a CAR molecule or comprises a nucleic acid encoding a CAR molecule.

66. 65. The reaction mixture of claim 64, wherein the BTK inhibitor is selected from ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13.

67. 65. The reaction mixture of claim 64, further comprising cancer cells.

68. A reaction mixture comprising a population of immune effector cells and a CAR molecule or a nucleic acid encoding a CAR molecule, wherein the immune effector cells comprise covalently inactivated ITK.

69. 69. The reaction mixture of claim 68, further comprising cancer cells.

70. 69. The reaction mixture of claim 68, wherein the cancer cells comprise covalently inactivated BTK.

71. 1. A composition comprising a cell expressing a CAR molecule that binds to CD19 ("CAR19-expressing cell") and one or more kinase inhibitors, wherein the kinase inhibitor is selected from a Bruton's tyrosine kinase (BTK) inhibitor, a cyclin-dependent kinase 4 (CDK4) inhibitor, an mTOR inhibitor, or a mitogen-activated protein kinase-interacting kinase (MNK) inhibitor.

72. 72. The composition of claim 71, wherein the CAR19-expressing cells and the one or more kinase inhibitors are present in a single dose form or in two or more dose forms.

73. 73. A composition according to claim 71 or 72 for use as a medicament.

74. 73. The composition of claim 71 or 72 for use in treating a disease associated with CD19 expression.

75. 75. The use, method or composition of any of claims 1 to 74, wherein the CAR19-expressing cell is a human immune effector cell (e.g., a human T cell or a human NK cell) or population of cells.

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